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

Abstract

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 α₂-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.

Introduction

The NG108-15 neuroblastoma×glioma hybrid cell line has been previously shown to express imidazole binding sites (Feinland et al., 1988) and the putative endogenous ligand for imidazoline binding sites, known as clonidine-displacing substance (CDS; Ernsberger et al., 1989).

Crude CDS has been extracted from several tissues and sera to date, including the bovine brain Atlas and Burstein, 1984, Parker et al., 1999a, bovine lung Singh et al., 1995, Parker et al., 1999a, bovine adrenal glands (Parker et al., 1999a), rat brain (Chan et al., 1997), rat serum, adrenal gland, gastric fundus and heart Hensley et al., 1989, Meeley et al., 1992, human serum Kreisberg et al., 1987, Dontenwill et al., 1993 and human cerebrospinal fluid (Goldberg-Stern et al., 1993). Currently, the chemical nature of CDS is undefined, although several substances have been postulated. Agmatine displays the ability to bind to imidazoline sites, but lacks the functionality associated with classical CDS, as defined by Atlas (1994). Prell et al. (1998) have also reported preliminary data suggesting that imidazoleacetic acid-ribotide is also an endogenous I₁-site ligand. Finally Grigg et al. (1998) have partially purified a CDS. Collectively, none of these compounds appear to resemble the classical CDS, based upon physiochemical characteristics, but these data illustrate the potential for several CDS molecules to exist endogenously.

Since CDS has been identified from many tissues and species, finding an optimum source from which to elucidate the structure of this substance is paramount. The aim of this particular study was therefore, to extract crude CDS from the NG108-15 cells and purify this CDS further via reverse phase-HPLC (RP-HPLC).