Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027819 (neuroblastoma)
27,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The murine neuroblastoma N1E-115 cell line possesses a high density of angiotensin II (AngII) receptors that can be solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. These solubilized binding sites exhibited high affinity for CGP-42112A and not Losartan, indicating that they were of the AT2 subtype. However, displacement of 125I-AngII with the AT2 nonpeptide antagonist PD-123319 resulted in a biphasic curve, suggesting heterogeneity of the AT2 receptor population in N1E-115 cells. In support of this view, separation of two receptor populations was accomplished with heparin-Sepharose chromatography. More specifically, three distinct protein peaks eluted from the heparin-Sepharose column, two of which bound 125I-AngII with high affinity and saturability. One of these binding peaks (peak I) eluted rapidly and represented approximately 80% of the total binding activity, whereas the remaining binding activity was contained within a second peak (peak III) that required the addition of 1.5 M NaCl for its complete elution. Pharmacological analysis revealed that both peaks of binding activity were exclusively AT2 receptors insofar as they exhibited high affinity for CGP-42112A and little or no affinity for the AT1-selective antagonist Losartan. However, whereas the nonpeptidic AT2-selective antagonist PD-123319 completely displaced the binding of 125I-AngII from peak I in a monophasic fashion (IC50 = 9.1 +/- 4.1 nM; mean +/- SEM; n = 3), PD-123319 was much less effective in displacing 125I-AngII from peak III (IC50 = 196 +/- 27 nM; mean +/- SEM; n = 3). Treatment of individual peaks with the reducing agent dithiothreitol caused a large increase in 125I-AngII specific binding in peak III, whereas a decrease in binding was observed in peak I. Moreover, GTP gamma S significantly reduced high-affinity agonist binding in peak I but not peak III, further suggesting heterogeneity in the AT2 receptor family. Finally, immunoblotting studies with polyclonal antisera raised against peak I specifically detected two proteins of 110 and 66 kDa, as is true in crude solubilized membranes, whereas no immunospecific proteins were detected in peak III. These same antisera immunoprecipitated 125I-AngII binding activity in peak I but were ineffective in peak III. Collectively, these results suggest that heparin-Sepharose chromatography can efficiently separate two pharmacologically, biochemically and immunologically distinct populations of AT2 receptors.
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PMID:Biochemical characterization of two distinct angiotensin AT2 receptor populations in murine neuroblastoma N1E-115 cells. 818 20

Receptors with seven transmembrane domains (7TM) constitute a large family of structurally and functionally related proteins which respond to various types of ligands. We describe here the cloning and expression of a human 7TM receptor, denoted hFB22 (human Fetal Brain 22), which is the homologue (92% amino acid identity) of a bovine receptor (LCR1) reported by others to bind neuropeptide Y (NPY) with a pharmacological profile of the Y3 receptor subtype. However, upon expression in COS1 (confirmed by Northern analysis), COS7 or CHO-K1 cells, the hFB22 receptor did not confer specific 125I-Bolton-Hunter-NPY, 3H-propionyl-NPY or 125I-peptide YY (PYY) binding sites, in either intact cells or in membrane preparations. Similarly, cells transfected with the corresponding bovine clone (LCR1) did not show specific NPY/PYY binding exceeding that resulting from endogenous binding sites; mock-transfected COS7 cells, used frequently for heterologous expression of receptors, were found to have endogenous specific 125I-NPY binding sites (Bmax = 112 fmol/mg protein; Kd = 0.25 nM). Moreover, the hFB22 transfected cells, when compared to control transfected cells, did not display de novo NPY- or PYY-induced second messenger responses, i.e., (1) inhibition of forskolin-stimulated cAMP accumulation or (2) 45Ca2+ influx. The presence of hFB22 mRNA was detected in several human neuroblastoma cell lines, none of which was found to express Y3-like NPY binding sites. hFB22 displays 39% amino acid sequence identity (in the transmembrane regions) to the human interleukin-8 receptor, and 32-36% amino acid identity to the human receptors of angiotensin II, bradykinin, and n-formylpeptide, but only 23% amino acid identity to the previously described human NPY/PYY receptor of the Y1 receptor subtype. Our results show that hFB22 and LCR1 do not encode NPY receptors, and their true ligand(s) remains to be identified.
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PMID:A proposed bovine neuropeptide Y (NPY) receptor cDNA clone, or its human homologue, confers neither NPY binding sites nor NPY responsiveness on transfected cells. 823 9

The murine neuroblastoma N1E-115 cell line possesses type 1 and type 2 angiotensin II (AngII) receptor subtypes. In vitro differentiation of these cells substantially increases the density of the AT2-receptor subtype, whereas the density of the AT1 receptors remains unchanged. In the present study, we report that the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) selectively solubilized AT2 receptors from N1E-115 cell membranes and that these receptors could be purified further to near homogeneity by affinity chromatography. More specifically, the presence of an agonist (AngII) during affinity purification of AT2 receptors resulted in the elution of high (110-kDa) and low (66-kDa) molecular mass proteins as determined by gel electrophoresis under nonreducing conditions. In contrast, when the nonselective antagonist Sar1,Ile8-AngII was used during purification, only the lower 66-kDa protein was observed. Affinity purification in the presence of the peptide and nonpeptide AT2-receptor antagonists CGP42112A and PD123319 also resulted in elution of the same 66-kDa protein, but unlike that in the presence of Sar1,Ile8-AngII, some of the high molecular weight site was observed as well. On the other hand, Losartan, an AT1-receptor antagonist, was completely ineffective in eluting any AngII receptors from the affinity column, further confirming their AT2 identity. After agonist elution, the 110-kDa band dissociated into two low molecular mass bands of 66 kDa and 54 kDa when sodium dodecyl sulfate-gel electrophoresis was run under reducing conditions.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Affinity purification of angiotensin type 2 receptors from N1E-115 cells: evidence for agonist-induced formation of multimeric complexes. 826 26

Murine neuroblastoma N1E-115 cells are a useful system in which to study neuronal angiotensin II (AngII) receptors. N1E-115 cells possess both type 1 (AT1) and type 2 (AT2) AngII receptor subtypes, as does mammalian brain. AT2 receptors in brain or N1E-115 cells can be solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. In the present study, heparin-Sepharose chromatography was used to partially purify solubilized N1E-115 membranes to produce an enriched population of AT2 receptors. Subsequently, an eluted peak, containing the majority of AT2 binding activity, was used as an immunogen in the development of protein-directed polyclonal antibodies. The antibodies specifically detected immunoreactive proteins of approximately 110 and 66 kDa in both solubilized N1E-115 cells, as well as the original protein material that eluted from the heparin-Sepharose column, whereas no such immunoreactivity was detected in a kidney epithelial cell line that lacks any specific 125I-labeled AngII (125I-AngII) binding activity. Moreover, the antibodies immunoreacted with affinity-purified AT2 receptors. These antibodies were also able to immunoprecipitate AT2 receptors from solubilized N1E-115 cells, as revealed by the pharmacologic profile of 125I-AngII binding to the precipitated protein. Similarly, the antibodies were able to immunoprecipitate a 66-kDa protein that had been covalently crosslinked with 125I-AngII by use of the homobifunctional crosslinker dithiobis(succinimidyl propionate). Collectively, these results demonstrate the development of a specific AT2 receptor antibody that may be used to further characterize this receptor subtype at both the cellular and molecular levels.
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PMID:Development of polyclonal antibodies against angiotensin type 2 receptors. 836 47

Murine neuroblastoma N1E-115 cells possess membranous receptors for the octapeptide angiotensin II (AngII) whose density is substantially increased by in vitro differentiation. Incubation of differentiated N1E-115 cells with AngII produced a rapid decrease in receptor density, but did not alter the affinity of these receptors for either 125I-AngII or the high-affinity antagonist 125I-[Sarc1,Ile8]-AngII. This apparent down-regulation was dose related with an ED50 of 1 nM, and maximal decreases of approximately 90% were obtained with 100 nM AngII. Receptor loss from differentiated cell membranes was unaffected by incubations of membranes obtained from agonist-exposed cells with non-hydrolyzable analogues of GTP for 60 min at 37 degrees C to ensure dissociation of the ligand. Partial loss of AngII receptors was apparent within 5 min of agonist exposure, whereas maximal declines were not observed until 30 min. This temporal pattern resulted from a preferential decrease in the AT1 receptor subtype during the first 5 min, followed by a decline in both AT1 and AT2 receptors with longer periods of agonist exposure. The loss of membranous receptors was reversible with partial recovery observed after 4 h, and with nearly full recovery observed 18 h after exposure of the cells to AngII. However, the long-term recovery of receptor density was blocked by the protein synthesis inhibitor, cycloheximide. The heptapeptide angiotensin III produced a similar down-regulation of receptors, and the high-affinity antagonist [Sarc1,Thr8]-AngII blocked agonist-induced down-regulation. Finally, the apparent loss of cell surface AngII receptors decreased the ability of AngII to stimulate cyclic GMP production within intact N1E-115 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Down-regulation of angiotensin II receptor subtypes and desensitization of cyclic GMP production in neuroblastoma N1E-115 cells. 838 Jan 93

This laboratory has previously reported that angiotensin II is a growth factor for human SH-SY5Y neuroblastoma cells, and that a variety of converting enzyme inhibitors and angiotensin II antagonists reduce thymidine incorporation into the DNA of these cells. In the present study, insulin, at 5 micrograms/mL, was found to stimulate thymidine incorporation in SH-SY5Y cells. The insulin effect was only partially inhibited by the converting enzyme inhibitors enalapril, quinapril, and quinaprilat, whereas it was markedly or totally blunted by the angiotensin II antagonists DuP753 and PD123177. In additional studies, IGF-1 (50 ng/mL) significantly stimulated thymidine incorporation into these cells in a fashion indistinguishable from that of insulin. Taken together, these studies are consistent with the suggestion that insulin at high concentrations and IGF at low concentrations enhance the proliferative response of these cells to angiotensin II. The differential effects of converting enzyme inhibition and angiotensin II antagonism on cell proliferation could be explained if converting enzyme inhibition results in low, but effective, levels of angiotensin II in the culture medium, whereas the angiotensin II antagonists effectively block angiotensin II at its receptor. Finally, in this system, both the AT1 receptor blocking agent DuP 753 and the AT2 receptor blocking agent PD123177 appear to be effective.
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PMID:The interaction of insulin and angiotensin II on the regulation of human neuroblastoma cell growth. 846 92

A stable cell line expressing the angiotensin II (AII) receptor has been obtained by transfecting the human neuroblastoma SH-SY5Y with the plasmid pCEP4 containing the entire coding region of the rat angiotensin AII receptor AT1A. Angiotensin II (AII; 1-100 nM) evokes the release of [3H]noradrenaline ([3H]NA) in this cell line. Pretreatment with 100 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) enhances the AII-evoked release of [3H]NA approximately two-fold. Removal of extracellular Ca2+ ([Ca2+]o) decreases 100 nM AII-evoked release of [3H]NA by over 50% both in the presence and absence of TPA. AII increases intracellular Ca2+ ([Ca2+]i) in this cell line which is consistent with the AT1A receptor being coupled to phospholipase C. Pretreatment with 100 nM TPA for 8 min attenuated the effect of AII on [Ca2+]i. The effects of AT1A receptor stimulation are therefore regulated differently in this cell line by activation of protein kinase C (PKC). Thus a useful cell line has been obtained from the human neuroblastoma SH-SY5Y in which to study at the molecular level the mechanism(s) by which AII regulates NA release.
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PMID:The effect of the angiotensin II (AT1A) receptor stably transfected into human neuroblastoma SH-SY5Y cells on noradrenaline release and changes in intracellular calcium. 858 37

Differentiation of NG108-15 neuroblastoma cells following exposure to either 1.5% dimethyl sulfoxide (DMSO)/0.5% fetal bovine serum (FBS) or serum starvation resulted in significant differences in angiotensin (AT) receptor levels and the AT1/AT2 receptor ratio. When NG108 cells were differentiated for 4 days with DMSO/low serum, the number of AT binding sites increased 30-fold compared with the binding levels on undifferentiated (blast) cells. However, cells differentiated by serum starvation for 4 or 14 days resulted in only a modest 2.5- and fivefold increase in AT receptor levels, respectively, over the levels seen with the undifferentiated cells. KD values for all treatment conditions were not significantly different (0.71 +/- 0.11 nM, p = 0.06). Using the AT1 and AT2 isoform-specific receptor antagonists losartan and PD123319, the relative numbers of AT receptor subtypes on undifferentiated and differentiated cells were determined by competitive inhibition against 125I-[Sar1,Ile8]-angiotensin II (sarile). A majority of the AT receptors on undifferentiated NG108 cells were the AT1 subtype (AT1/AT2 receptor ratio of 8:3). Differentiation by serum starvation and DMSO/low serum treatment resulted in fivefold and 30-fold increases in AT receptor levels, respectively, compared with the levels seen with the undifferentiated cells. Although serum starvation increased the total number of AT1 and AT2 receptors, it did not significantly alter the AT1/AT2 receptor ratio. In contrast, differentiation with DMSO/low serum both increased the total number of AT1 and AT2 receptors and reversed the AT1/AT2 receptor ratio (1:3). The increase in AT receptors following differentiation with DMSO/low serum for 4 days was largely accounted for by an 80-fold increase in the AT2 receptor level. Previous studies by Tallant at al. (1991) and Bryson et al. (1992) reported increased AT2 receptor expression following neuroblastoma differentiation with dibutyryl cyclic AMP and DMSO/low serum, respectively, and suggested a role for the AT2 receptor in neuronal differentiation. In the present study, we have extended these earlier observations by demonstrating that the method of differentiation significantly affects both the AT receptor level and the ratio of AT1 to AT2 receptor expression. Finally, our findings indicate that the AT2 receptor is expressed as a consequence of neuronal maturation and dose not mediate morphological differentiation.
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PMID:Differentiation of NG108-15 neuroblastoma cells by serum starvation or dimethyl sulfoxide results in marked differences in angiotensin II receptor subtype expression. 876 61

All the angiotensin peptides originate from angiotensinogen, a glycoprotein synthesized by several tissues, including the brain and the anterior pituitary. In the rat, immunohistochemistry has been used to localize angiotensinogen in gonadotropes and in uncharacterized cells surrounding sinusoids. Both cell types are capable of secreting angiotensinogen in cell culture; only the gonadotropes contain angiotensin II (AngII) and are capable of secreting it in culture. It has been asserted that the perisinusoidal cells are the only source of angiotensinogen for the generation of AngII by gonadotropes. Our current data favor the existence of a complete intracellular renin-angiotensin system (RAS) in gonadotropes and a separate extracellular system which utilizes the high concentration of angiotensinogen from perisinusoidal cells. Furthermore, we postulate that gonadotrope AngII serves mainly reproductive functions, while the proximity of angiotensinogen-secreting cells to folliculostellate cells, and their access to the intercellular sinusoidal and follicular spaces, places the extracellular RAS in a strategic position to affect pituitary growth and the mediation of acute-phase immune responses. In the rat brain, angiotensinogen is expressed by the 16-18th day of fetal life and by areas generally concerned with vasopressor, electrolyte, and fluid homeostasis. Antisense deoxyoligonucleotides to angiotensinogen mRNA lower blood pressure in hypertensive rats and inhibit in vitro growth of neuroblastoma cells, indicating a significant role for angiotensinogen in mitogenic and homeostatic functions. It is commonly agreed that astrocytes express angiotensinogen. Neuronal angiotensinogen has also been demonstrated by immunohistochemistry, as a secretion from neuronal cell cultures, and by reverse-transcriptase polymerase chain reaction. The fate of secreted astrocytic and neuronal angiotensinogen remains obscure. Angiotensinogen is regulated in a tissue-specific manner with smaller or absent responses observed for brain tissue. By using astrocyte and neuronal cultures the actions on angiotensinogen production of growth hormone, IGF-1, inflammatory lipopolysaccharide, and phorbol ester have been examined. Recent observations show that angiotensinogen is regulated positively or negatively by glucocorticoids and that a positive synergism between cAMP and glucocorticoids exists. On the basis of analogous systems for other proteins, a scheme involving glucocorticoid receptors, CREB, and AP-1 transcription factors is formulated to explain glucocorticoid-cAMP interactions. These transcriptional interactions may form a significant functional link between the RAS and adrenergic mechanisms.
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PMID:Novel perspectives on pituitary and brain angiotensinogen. 910 Dec 59

Homology-based PCR was used to isolate angiotensin II type 2 (AT2) receptor cDNA from murine neuroblastoma N1E-115 cells. Despite subtle differences in the nucleotide sequence (the N1E-115 clone coded for Phe133 as TTC and Gln326 as CAG; base substitutions are in bold-italics), the AT2 receptor protein was identical to other reported murine AT2 clones. When transfected into COS-1 cells, the expressed AT2 receptor displayed high affinity for AngII and for AT2-selective compounds, GTP gamma S-insensitive agonist binding and enhanced agonist binding by dithiothreitol. Previously, we have demonstrated that N1E-115 cells possess two distinct subpopulations of AT2 receptors, defined as peak I and peak III receptors, that can be separated by heparin-sepharose chromatography. The two subpopulations differ pharmacologically, biochemically and immunologically. The binding properties of the cloned AT2 receptor closely resembled that of peak III receptors. Moreover, antisera raised against peak I AT2 receptors failed to immunoreact to either peak III receptors or cloned AT2 receptors expressed in COS-1 cells. Collectively, these data suggest that the cloned AT2 receptor is identical to peak III receptors from N1E-115 cells and that a novel AT2 receptor (peak I) remains to be cloned.
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PMID:Cloning and expression of angiotensin II type 2 (AT2) receptors from murine neuroblastoma N1E-115 cells: evidence for AT2 receptor heterogeneity. 910 76


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