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A good in vitro model within which to investigate molecular interactions between feeding relevant neuropeptide systems has been lacking. Consequently, we began using reverse transcriptase-polymerase chain reaction (RT-PCR) to screen various neuronal cell lines for the presence of feeding relevant neuropeptides and receptors. N1E-115 murine neuroblastoma cells have emerged as an attractive candidate for further analysis because they contain mRNA for a variety of key systems implicated in the regulation of energy homeostasis., (Copyright 2002 Elsevier Science B.V.)

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.

Three human neuroblastoma cell lines were shown to have markedly different contents of catecholamines and serotonin. Two of the cell lines (CHP-134 and IMR-5) have higher levels of dopamine and its metabolites, while CHP-404 cells have higher levels of serotonin and its metabolites. Each cell line responded to the addition of D,L-2-amino-5-phosphonovalerate, an agent which increases plasma membrane permeability to Ca2+ (Pastuszko and Wilson, 1988; with striking changes in the metabolism of the neurotransmitters. These changes were dependent on the extracellular calcium concentration and include activation of dopamine synthesis (tyrosine hydroxylase), increased levels of dihydroxyphenylacetic acid and increased formation of N-methylated dopamine derivatives. Catabolism of serotonin to 5-hydroxyindole acetic acid was inhibited while that to 5-hydroxytryptophol was stimulated. These data clearly identify several important sites for regulation of neurotransmitter metabolism by calcium. The mechanisms, direct or indirect, by which the enzyme activities are modulated by calcium remain to be established.