Human astrocytoma cells (U-87 MG) exhibit a specific substance P binding site with the characteristics of an NK-1 receptor.
To investigate substance P (SP) receptors on an established human astrocytoma cell line (U-87 MG), [3H][Sar9,Met(O2)11]-SP, a selective SP receptor agonist, was used to identify and characterize the cell membrane binding sites for SP. SP receptor mRNA was examined by solution hybridization analysis, and the existence of SP binding protein on the surface of membranes was evaluated by flow cytometry using an anti-SP binding protein antibody. In U-87 MG and U-373 MG RNA preparations, transcripts were identified that corresponded to both mature and partially spliced receptor forms. In U-87 MG cell membrane-enriched preparations, the binding of [3H][Sar9,Met(O2)11]-SP was found to be time and cell number dependent, specific, saturable, and of high affinity. Equilibrium binding analysis revealed a single class of binding sites with an apparent KD of 1.15 +/- 0.15 nM and a Bmax of 108 +/- 9.8 fmol/mg of protein. [3H][Sar9, Met(O2)11]-SP binding was basically not influenced by addition of mono (Na+, Li+) or divalent (Mg2+, Mn2+, Ca2+) cations; only high doses of divalent cations decreased the binding. GTP and guanylyl-5'-imidodiphosphate, but not GDP and GMP, reduced the Bmax without changing the affinity of [3H][Sar9,Met(O2)11]-SP. We also examined the effects of pretreatment with three lectins [concanavalin A (con A), wheat germ agglutinin (WGA), and Lens culinaris agglutinin (LCA)] to determine the nature of carbohydrate chains on the U-87 MG cell. Of three lectins analyzed for effects on agonist binding, WGA and LCA had an inhibitory effect, whereas con A was ineffective. These results suggest that SP receptors on the human astrocytoma cell line U-87 MG have either a biantennary complex-type or a high mannose-type of carbohydrate chain and may be regulated by GTP-binding protein(s).
Ogo
H
H
Department of Pharmacology, Hiroshima University School of Medicine, Japan.
Kuroyanagi
N
N
Inoue
A
A
Nishio
H
H
Hirai
Y
Y
Akiyama
M
M
DiMaggio
D A
DA
Krause
J E
JE
Nakata
Y
Y
D001254:Astrocytoma; D001665:Binding Sites; D001667:Binding, Competitive; D002414:Cations, Monovalent / Q000494:pharmacology; D002460:Cell Line; D005091:Exons; D005909:Glioblastoma; D006150:Guanine Nucleotides / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D037102:Lectins / Q000494:pharmacology; D016372:RNA, Antisense; D018075:RNA, Complementary; D012333:RNA, Messenger / Q000378:metabolism; D018040:Receptors, Neurokinin-1 / Q000819:agonists / Q000096:biosynthesis / Q000378:metabolism*; D013373:Substance P / Q000031:analogs & derivatives / Q000378:metabolism*; D014316:Tritium; D014407:Tumor Cells, Cultured
D002414:Cations, Monovalent; D006150:Guanine Nucleotides; D037102:Lectins; D016372:RNA, Antisense; D018075:RNA, Complementary; D012333:RNA, Messenger; D018040:Receptors, Neurokinin-1; D014316:Tritium; C062618:substance P, Sar(9)-Met(O2)(11)-; D013373:Substance P; D006160:Guanosine Triphosphate