Isolation of RP-HPLC pure clonidine-displacing substance from NG108-15 cells

Eur J Pharmacol. 2000 Jan 3;387(1):27-30. doi: 10.1016/s0014-2999(99)00805-5.


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.

MeSH terms

  • Animals
  • Binding, Competitive
  • Brain / metabolism
  • Chromatography, High Pressure Liquid / methods
  • Clonidine / analogs & derivatives*
  • Clonidine / analysis
  • Clonidine / isolation & purification
  • Clonidine / metabolism
  • Hybrid Cells
  • Membranes / metabolism
  • Radioligand Assay
  • Rats
  • Receptors, Adrenergic, alpha-2 / metabolism
  • Tumor Cells, Cultured


  • Receptors, Adrenergic, alpha-2
  • clonidine-displacing substance
  • Clonidine