Characterization of a Human NK₁ Tachykinin Receptor in the Astrocytoma Cell Line U 373 MG

Abstract

The human NK1 tachykinin receptor in the astrocytoma cell line U 373 MG was characterized using selective agonists and antagonists described for this receptor in the rat. Specific [3H]substance P binding sites were present on cell homogenates, whereas [3H]neurokinin A or [3H]-senktide binding sites were absent. The binding was saturable and reversible. The binding of [3H]substance P was inhibited by very low concentrations of [L-Pro9]substance P and [Sar9,Met(O2)11]substance P; septide was approximately 1,000-fold less potent. The most potent peptide antagonist was trans-4-hydroxy-1-(1H-indol-3-ylcarbonyl)-L-prolyl-N-methyl-N-(phe nylmethyl)-L- tyrosineamide. The rank order of potency for the nonpeptide antagonists was (S,S)-CP 96,345 > (+/-)-CP 96,345 > (+/-)-2-chlorobenzylquinuclidinone > (R,R)-CP 96,345 > RP 67580 > RP 68651. In [3H]-inositol-labeled cells, substance P stimulated phosphatidylinositol turnover. A good correlation was found when the abilities of NK1 receptor agonists for stimulating inositol phosphate production and for inhibiting [3H]substance P binding were compared. Similarly, the binding and functional assays were well correlated for the antagonists. As a result of its high sensitivity and selectivity, the U 373 MG cell line thus appears an excellent tool for investigating the pharmacology of the human NK1 receptor.

The Neuropeptide Substance P Activates p38 Mitogen-Activated Protein Kinase Resulting in IL-6 Expression Independently from NF-κB

“…As a cellular system, we used the astrocytoma cell line U373 MG which shares several characteristics with primary rat astrocytes (16,26,27). U373 MG astrocytoma cells express functional high-affinity NK-1 receptors (16,24,25) and are able to express IL-6 in response to SP treatment (7,28). Therefore, this cell line is widely used as an in vitro model for the analysis of NK-1 receptor function.…”

“…The cells have been reported to express functional NK-1 receptors (24,25). To investigate the effects of SP on the activation of p38 and p42/44 MAPK, cells were treated with 10 nM SP for different time periods.…”

Anti-tumour activity of tachykinin NK1 receptor antagonists on human glioma U373 MG xenograft

“…interleukin (IL)-6, IL-8, transforming growth factor-β, leukaemia inhibitory factor, granulocyte-macrophage colony stimulating factor) which are thought to be relevant for glioma progression (Gitter et al, 1994;Palma et al, 1995;Palma and Manzini, 1998). In a large number of these studies, the human astrocytoma grade III U373 MG cell line, which expresses a high level of functional tachykinin NK 1 receptor (Lee et al, 1992;Palma et al, 1999b) but not tachykinin NK 2 or NK 3 receptors (Heuillet et al, 1993), were used.The role of SP in glioma activities may be ascribed to a pathological use of normal signal transduction pathways occurring in reactive astrocytes. In fact, SP activates phospholipase C and stimulates the release of IL-6 and prostaglandin E 2 from human fetal astrocytes in culture (Palma et al, 1997), and there is evidence for an up-regulation of this receptor by reactive proliferating astrocytes after transection of the optic nerve (Mantyh et al, 1989).…”

“…interleukin (IL)-6, IL-8, transforming growth factor-β, leukaemia inhibitory factor, granulocyte-macrophage colony stimulating factor) which are thought to be relevant for glioma progression (Gitter et al, 1994;Palma et al, 1995;Palma and Manzini, 1998). In a large number of these studies, the human astrocytoma grade III U373 MG cell line, which expresses a high level of functional tachykinin NK 1 receptor (Lee et al, 1992;Palma et al, 1999b) but not tachykinin NK 2 or NK 3 receptors (Heuillet et al, 1993), were used.…”

“…In a large number of these studies, the human astrocytoma grade III U373 MG cell line, which expresses a high level of functional tachykinin NK 1 receptor (Lee et al, 1992;Palma et al, 1999b) but not tachykinin NK 2 or NK 3 receptors (Heuillet et al, 1993), were used.…”