angiotensin-iii and Neuroblastoma

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

The effects of angiotensin II (AII) and related peptides on the mobilization of internal Ca2+ were studied in a subclone of NG 108-15 cells. The subclone, C1, was prepared by fluorescence-activated cell cloning using a rapid response kinetics and a large response magnitude following stimulation by AII as the selection criteria. Angiotensin I, AII, and angiotensin III (AIII) stimulated Ca2+ mobilization in the C1 cells in a concentration-dependent manner (1 nM-100 microM), yielding EC50 values of 437 +/- 80 nM (n = 4; slope = 1.6 +/- 0.3), 57 +/- 8 nM (n = 12; slope = 1.5 +/- 0.3), and 36 +/- 5 nM (n = 7; slope = 1.4 +/- 0.3), respectively. AIII was significantly more potent than AII (p less than 0.05). In contrast, Des-Phe8-AII, AII-hexapeptide (AII 3-8), and p-NH2-Phe6-AII (1-10 microM) were inactive as agonists. Although the effects of AII and AIII in C1 and parent NG108-15 cells were totally inhibited by the AT1 receptor-selective nonpeptide antagonist, DUP-753 (0.3-1 microM), the AT2-selective antagonists, EXP-655 and CGP42112A (1-10 microM), failed to block the effects of AII. DUP-753 (0.3-100 nM) produced dextral shifts of the AII-induced concentration-response curves and yielded an estimated affinity constant (pA2) of 8.5 +/- 0.2 (n = 16) using single-point analysis involving different concentrations of DUP-753. These data compared well with those obtained for the inhibition of AII-induced aortic contractions by DUP-753 (pA2 = 8.5) reported previously by others.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Angiotensin I; Angiotensin II; Angiotensin III; Angiotensin Receptor Antagonists; Animals; Biphenyl Compounds; Calcium; Imidazoles; Intracellular Membranes; Losartan; Neuroblastoma; Receptors, Angiotensin; Tetrazoles; Tumor Cells, Cultured

Neuroblastoma x glioma hybrid cells (NG108-15), differentiated by treatment with 1.5% dimethyl sulfoxide (DMSO) and 0.5% fetal bovine serum, were used to measure the effect of angiotensin II and III (ANG II and ANG III) on the generation of inositol polyphosphates. ANG II increased the synthesis of inositol monophosphates (IP1), inositol diphosphates (IP2), and inositol trisphosphates (IP3) with maximal responses observed at 300, 120, and 30 sec, respectively. The percent increases above basal values at the maximal responses were 140% +/- 9% (IP1), 142% +/- 4% (IP2), and 132% +/- 4% (IP3). This effect was not attenuated by pretreatment of the cells with pertussis toxin. Furthermore, both ANG II and ANG III increased the production of inositol polyphosphates in a dose-dependent manner with ED50 values of 145 nM and 11 nM, respectively. We conclude that differentiated NG108-15 cells express an ANG III selective receptor that mediates phosphatidylinositol breakdown through a pertussis toxin insensitive G-protein.

Topics: Angiotensin II; Angiotensin III; Animals; Cell Differentiation; Cell Line; Glioma; Hybrid Cells; Inositol; Inositol Phosphates; Kinetics; Mice; Neuroblastoma; Rats

The murine neuroblastoma N1E-115 cell line contains binding sites for the angiotensin II (Ang II) receptor antagonist 125I-[Sarc1,Ile8]-Ang II (125I-SARILE). Binding of 125I-SARILE to N1E-115 membranes was rapid, reversible, and specific for Ang II-related peptides. The rank order potency of 125I-SARILE binding was the following: [Sarc1]-Ang II = [Sarc1,Ile8]-Ang II greater than Ang II greater than Ang III = [Sarc1,Thr8]-Ang II much greater than Ang I. Scatchard analysis of membranes prepared from confluent monolayers revealed a homogenous population of high affinity (KD = 383 +/- 60 pM) binding sites with a Bmax of 25.4 +/- 1.6 fmol/mg of protein. Moreover, the density, but not the affinity, of the binding sites increased as the cells progressed from logarithmic to stationary growth in culture. Finally, agonist, but not antagonist, binding to N1E-115 cells was regulated by guanine nucleotides. Collectively, these results suggest that the murine neuroblastoma N1E-115 cell line may provide a useful model in which to investigate the signal transduction mechanisms utilized by neuronal Ang II receptors.

Topics: 1-Sarcosine-8-Isoleucine Angiotensin II; Angiotensin I; Angiotensin II; Angiotensin III; Angiotensin Receptor Antagonists; Animals; Binding, Competitive; Cell Division; Cell Membrane; Guanylyl Imidodiphosphate; Kinetics; Mice; Neuroblastoma; Receptors, Angiotensin; Tumor Cells, Cultured

Topics: Angiotensin I; Angiotensin II; Angiotensin III; Angiotensins; Animals; Clone Cells; Cyclic GMP; Mice; Neuroblastoma; Neurons; Receptors, Angiotensin