Isolation of RP-HPLC pure clonidine-displacing substance from NG108-15 cells.
A crude extract of clonidine-displacing substance (CDS) has previously been extracted from the NG108-15 cell line. This study aimed to purify CDS extracted from this cell line further, by the technique of reverse phase-HPLC (RP-HPLC), and subsequently determine whether this refined CDS bears any similarity to CDS's extracted from other tissues. Crude CDS was extracted from NG108-cells and fractionated by RP-HPLC eluting with a linear gradient of methanol (5-65%; 1 ml min(-1) flow rate) over 50 min., and collected at 1 min. intervals. The pharmacological activities of the CDS fractions were determined by their abilities to displace bound [3H]clonidine to alpha(2)-adrenoceptors in rat brain membranes. RP-HPLC analysis of CDS revealed a pharmacologically active fraction distinct from agmatine, eluting at 24 min, corresponding to an absorbance peak observed at this time. Collectively, these results confirmed that CDS was present in the NG108-15 cell line. However, the RP-HPLC analysis showed the pharmacological activity to elute at a more hydrophobic gradient than previously observed with CDS's extracted from bovine tissues. These results support the notion of the existence of several CDS's.
Parker
C A
CA
Psychopharmacology Unit, School of Medical Sciences, University Walk, University of Bristol, Bristol, UK.
Hudson
A L
AL
Nutt
D J
DJ
Dillon
M P
MP
Eglen
R M
RM
Crosby
J
J
D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003000:Clonidine / Q000031:analogs & derivatives* / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D006822:Hybrid Cells; D008566:Membranes / Q000378:metabolism; D011869:Radioligand Assay; D051381:Rats; D018341:Receptors, Adrenergic, alpha-2 / Q000378:metabolism; D014407:Tumor Cells, Cultured
D018341:Receptors, Adrenergic, alpha-2; C055140:clonidine-displacing substance; D003000:Clonidine