Functional characterization of neurokinin‐1 receptors on human U373MG astrocytoma cells

Eistetter, Harald; Mills, Ann; Brewster, Rachel; Alouani, Sami; Rambosson, Christine; Kawashima, Eric

doi:10.1002/glia.440060203

Cited by 50 publications

(39 citation statements)

References 35 publications

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“…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).…”

Section: Discussionmentioning

confidence: 99%

“…From molecular studies, it is well known that SP binds to a G proteincoupled receptor of the neurokinin (NK) receptor family and that SP has a preferential affinity to the NK-1 receptor subtype (13)(14)(15). Intracellularly, receptor binding is followed by phosphoinositide hydrolysis and calcium mobilization (16). In two previous studies, we have investigated how the SP receptor-elicited signals affect transcriptional activation and novel inflammatory gene expression (7,17).…”

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confidence: 99%

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

Fiebich

Schleicher

Butcher

et al. 2000

The Journal of Immunology

127

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Substance P (SP), a member of the tachykinin peptide family, is a major mediator of neuroimmunomodulatory activities and neurogenic inflammation within the central and peripheral nervous system. SP has been shown to induce the expression of proinflammatory cytokines such as IL-6, which might be implicated in the etiopathology of several human brain disorders. We showed in a previous study that nanomolar concentrations of SP triggered activation of NF-κB, a transcription factor involved in the control of cytokine expression. However, activation of NF-κB was not involved in SP-induced expression of IL-6. Here, we describe p38 mitogen-activated protein kinase (p38 MAPK) as a signal transduction component that operates independently from NF-κB activation and that mediates SP-induced IL-6 expression in the human astrocytoma cell line U373 MG. SP induced the phosphorylation of p38 MAPK within 10 min, and this activation persisted up to 30 min and was independent from p42/44 MAPKs and protein kinase C activation, which all are induced after stimulation with SP. As shown by EMSA, p38 MAPK was not involved in SP-induced activation of NF-κB. p38 MAPK, however, mediated SP-induced IL-6 expression as shown by the use of specific inhibitors of this kinase. Our results suggest that activation of p38 MAPK is an important component controlling neurogenic inflammation within the CNS independently from NF-κB. Drugs targeting this MAPK may therefore interfere with SP-correlated neuropsychiatric disorders and may represent a therapeutic approach in these disorders.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Fiebich

Schleicher

Butcher

et al. 2000

The Journal of Immunology

127

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“…Chinese hamster ovary (CHO) cells were infected on 24 well plates (0.5 -1 X 10' cells/well) with SFV-NK-1 virus stock and were grown for 10, 16 or 24 h. At the end of this time, the cells were analysed for ['H]substance P binding as described by Eistetter et al [36]. Nonspecific binding was determined in the presence of 1 pM substance P methyl ester.…”

Section: Radioligand-binding Assaysmentioning

confidence: 99%

High‐level Expression of the Human Neurokinin‐1 Receptor in Mammalian Cell Lines using the Semliki Forest Virus Expression System

Lundström¹,

Mills²,

Buell³

et al. 1994

European Journal of Biochemistry

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Human neurokinin-1 receptor cDNA was introduced into the pSFVl Semliki Forest virus (SFV) vector and the in vitro transcribed RNA was electroporated into BHK cells with pSFV-Helper RNA. This procedure resulted in the packaging of a high-titer SFV-NK-1 virus stock containing approximately 5 X lo9 infective units/ml. Infection of baby hamster kidney, COS-7, Chinese hamster ovary and human osteosarcoma cells yielded high levels of human neurokinin-1 receptor expression as assessed by [3H]~ub~tance P binding. The maximal receptor expression level obtained was 4X106 receptors/cell and studies of the post-infection time indicated that a high level of receptor expression was observed 10-24 h post-infection. The human neurokinin-1 receptor expressed in infected baby hamster kidney, COS-7 and Chinese hamster ovary cells was able to stimulate Ca2+ mobilization indicating functional coupling to guanine-nucleotide-binding proteins. The application of the Semliki Forest virus expression system will permit the rapid and efficient production of large quantities of receptor protein for both pharmacological and structural studies. [8, 91. Instrumental in our current understanding of the structure and function of this receptor has been its molecular cloning from a number of different species [2, 10, 111 and many reports have appeared on the membrane topology, ligand binding and interaction with G proteins [12-151 of this protein. However, further progress is being hampered by the lack of large quantitities of purified receptor for structural studies. Moreover, in order to elucidate in more detail the structure/function relationships, receptor mutants will need to be investigated. Earlier strategies for testing seven-transmembrane receptor function have included injection of in vitm synthesized mRNA into Xenopus laevis oocytes [16, 171, expression in bacteria [18, 191 and yeast [20, 211, and Correspondence to K. Lundstrom, Department of Molecular BiFax: +41 22 7946965. Abbreviations. BHK, baby hamster kidney; CHO, Chinese hamster ovary; DMEM, Dulbecco's modified Eagle's medium; Fura-2,

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“…The study has been performed in U373MG cells endogenously expressing the human NK 1 R (Eistetter et al, 1992). The experiments in vitro were designed to evaluate competitive and insurmountable NK 1 R interactions, and functional reversibility was tested after pretreatment of antagonist.…”

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confidence: 99%

Neurokinin 1 Receptor Antagonists: Correlation between in Vitro Receptor Interaction and in Vivo Efficacy

Lindström¹,

Mentzer²,

Påhlman³

et al. 2007

J Pharmacol Exp Ther

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We compared the neurokinin 1 receptor (NK 1 R) antagonists aprepitant, CP-99994 [(2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine], and ZD6021 [3-cyano-N-((2S)-2-(3,4-dichlorophenyl)-4-[4-[2-(methyl-(S)-sulfinyl)phenyl]piperidino]butyl)-N-methyl]napthamide]] with respect to receptor interactions and duration of efficacy in vivo. In Ca 2ϩ mobilization assays (fluorometric imaging plate reader), antagonists were applied to human U373MG cells simultaneously with or 2.5 min before substance P (SP). In reversibility studies, antagonists were present for 30 min before washing, and responses to SP were repeatedly measured afterward. The compounds were administered i.p. to gerbils, and the gerbil foot tap (GFT) response was monitored at various time points. The NK 1 R receptor occupancy for aprepitant was determined in striatal regions. Levels of compound in brain and plasma were measured. Antagonists were equipotent at human NK 1 R and acted competitively with SP. After preincubation, aprepitant and ZD6021 attenuated the maximal responses, whereas CP-99994 only shifted the SP concentration-response curve to the right. The inhibitory effect of CP-99994 was over within 30 min, whereas for ZD6021, 50% inhibition still persisted after 60 min. Aprepitant produced maximal inhibition lasting at least 60 min. CP-99994 (3 mol/kg) inhibited GFT by 100% 15 min after administration, but the effect declined rapidly together with brain levels thereafter. The efficacy of ZD6021 (10 mol/kg) lasted 4 h and correlated well with brain levels. Aprepitant (3 mol/kg) inhibited GFT and occupied striatal NK 1 R by 100% for Ͼ48 h despite that brain levels of compound were below the limit of detection after 24 h. Slow functional reversibility is associated with long-lasting in vivo efficacy of NK 1 R antagonists, whereas the efficacy of compounds with rapid reversibility is reflected by their pharmacokinetics.The neurokinins substance P (SP), neurokinin (NK) A (NKA), and NKB belong to the tachykinin peptide family (Severini et al., 2002). The tachykinin receptors are divided into three subtypes: NK 1 R, NK 2 R, and NK 3 R. The rank order of potency of the endogenous tachykinins are: for NK 1 R, SP Ն NKA Ͼ NKB; for NK 2 R, NKA Ͼ NKB Ͼ SP; and for NK 3 R, NKB Ͼ NKA Ͼ SP (for review, see Pennefather et al., 2004). Hemokinin-1 and endokinins A and B are relatively new mammalian members of the tachykinin family but appear to have similar receptor pharmacology as SP (Page, 2006). On the other hand, endokinins C and D have negligible affinity for known NK receptors (Page, 2006).Preclinical research has implicated especially the NK 1 R as being involved in several pathological disorders, including emesis, asthma, psychiatric disorders, gastrointestinal disorders, pain, migraine, inflammation, and urinary bladder disorders. This has led to the subsequent development of selective and potent NK 1 R antagonists (for recent review, see Quartara and Altamura, 2006). However, so far, only aprepitant has reached the market for treatment of chemothe...

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Functional characterization of neurokinin‐1 receptors on human U373MG astrocytoma cells

Cited by 50 publications

References 35 publications

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

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

High‐level Expression of the Human Neurokinin‐1 Receptor in Mammalian Cell Lines using the Semliki Forest Virus Expression System

Neurokinin 1 Receptor Antagonists: Correlation between in Vitro Receptor Interaction and in Vivo Efficacy

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