Dimethyl sulfoxide: an antagonist in scintillation proximity assay [(35)S]-GTPgammaS binding to rat 5-HT(6) receptor cloned in HEK-293 cells?
We have tested by [(35)S]-GTPgammaS binding the intrinsic activity of three full agonists (serotonin, 5-methoxytryptamine and 5-methoxy-2-methyl-N,N-dimethyltryptamine) on rat 5-HT(6) 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 [(35)S]-GTPgammaS 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 [(35)S]-GTPgammaS. In addition, DMSO did not affect [(3)H]-5HT binding or cAMP accumulation in cloned HEK-293 cells expressing rat 5-HT(6) 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 [(35)S]-GTPgammaS. DMSO does not affect basal signal, nor the GTPgammaS 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.
Mereghetti
Ilario
I
"Mario Negri" Institute for Pharmacological Research, via Eritrea 62, 20157 Milan, Italy.
Cagnotto
Alfredo
A
Mennini
Tiziana
T
D008735:5-Methoxytryptamine / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000187:drug effects / Q000378:metabolism; D001667:Binding, Competitive / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D004121:Dimethyl Sulfoxide / Q000737:chemistry*; D005063:Europium / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D009448:Neurochemistry / Q000379:methods*; D011869:Radioligand Assay / Q000379:methods*; D051381:Rats; D011985:Receptors, Serotonin / Q000378:metabolism*; D012588:Scintillation Counting / Q000379:methods*; D012701:Serotonin / Q000378:metabolism; D012995:Solubility / Q000187:drug effects; D013462:Sulfur Radioisotopes / Q000378:metabolism
D000906:Antibodies; D011985:Receptors, Serotonin; D013462:Sulfur Radioisotopes; C097216:serotonin 6 receptor; D012701:Serotonin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D008735:5-Methoxytryptamine; D005063:Europium; D006160:Guanosine Triphosphate; D000242:Cyclic AMP; D004121:Dimethyl Sulfoxide