Dimethyl sulfoxide: An antagonist in scintillation proximity assay [35S]-GTPγS binding to rat 5-HT6 receptor cloned in HEK-293 cells?

Ilario Mereghetti, Alfredo Cagnotto, Tiziana Mennini

Research output: Contribution to journalArticle

Abstract

We have tested by [35S]-GTPγS binding the intrinsic activity of three full agonists (serotonin, 5-methoxytryptamine and 5-methoxy-2-methyl-N,N-dimethyltryptamine) on rat 5-HT6 receptors cloned in HEK-293 cells, using the scintillation proximity assay. Serotonin and 5-methoxytryptamine are soluble in water, while the agonist 5-methoxy-2-methyl-N,N-dimethyltryptamine is soluble in dimethyl sulfoxide (DMSO). In [35S]-GTPγS binding 5-HT and 5-methoxytryptamine were able to increase basal binding, while 5-methoxy-2-methyl-N,N-dimethyltryptamine surprisingly showed an inverse agonist activity. So we have tested 5-HT and 5-methoxytryptamine in the presence of DMSO: in this condition the two agonists behaved as antagonists. This interfering effect of DMSO was not observed when GTP-europium filtration binding was used in place of scintillation proximity assay using [35S]-GTPγS. In addition, DMSO did not affect [3H]-5HT binding or cAMP accumulation in cloned HEK-293 cells expressing rat 5-HT6 receptors. In conclusion, we demonstrated that DMSO, the most common solvent used to dissolve compounds insoluble in water, interferes with the method of scintillation proximity assay using [35S]-GTPγS. DMSO does not affect basal signal, nor the GTPγS binding itself, as indicated by the experiments with GTP-europium. Therefore its interfering effect is likely to occur at the binding of antibodies in the scintillation proximity assay.

Original languageEnglish
Pages (from-to)251-255
Number of pages5
JournalJournal of Neuroscience Methods
Volume160
Issue number2
DOIs
Publication statusPublished - Mar 15 2007

Fingerprint

HEK293 Cells
Dimethyl Sulfoxide
5-Methoxytryptamine
N,N-Dimethyltryptamine
Europium
Serotonin
Guanosine Triphosphate
Serotonin Receptor Agonists
Water
serotonin 6 receptor
Antibodies

Keywords

  • 5-HT receptor
  • [H]-5HT binding
  • CAMP accumulation
  • DMSO
  • Europium
  • GTPγS binding
  • HEK-293 cells
  • Scintillation proximity assay

ASJC Scopus subject areas

  • Neuroscience(all)

Cite this

Dimethyl sulfoxide : An antagonist in scintillation proximity assay [35S]-GTPγS binding to rat 5-HT6 receptor cloned in HEK-293 cells? / Mereghetti, Ilario; Cagnotto, Alfredo; Mennini, Tiziana.

In: Journal of Neuroscience Methods, Vol. 160, No. 2, 15.03.2007, p. 251-255.

Research output: Contribution to journalArticle

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