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 extr acted from this cell line further by the technique of reverse phase-HPLC (R P-HPLC), and subsequently determine whether this refined CDS bears any simi larity 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 m ethanol (5-65%; 1 ml min(-1) flow rate) over 50 min., and collected at 1 mi n. intervals. The pharmacological activities of the CDS fractions were dete rmined by their abilities to displace bound [H-3]clonidine to alpha(2)-adre noceptors in mt brain membranes. RP-HPLC analysis of CDS revealed a pharmac ologically active fraction distinct from agmatine, eluting at 24 min, corre sponding to an absorbance peak observed at this time. Collectively, these r esults confirmed that CDS was present in the NG108-15 cell Line. However, t he RP-HPLC analysis showed the pharmacological activity to elute at a more hydrophobic gradient than previously observed with CDS's extracted from bov ine tissues. These results support the notion of the existence of several C DS's. (C) 2000 Elsevier Science B.V. All rights reserved.