Molecular and physiological properties of clonidine-displacing substance.

Abstract

An endogenous small molecular mass compound, termed clonidine-displacing substance (CDS), has been isolated and purified from bovine brain. The estimated level of CDS in bovine brain is 400-1,000 units/wet brain, with 1 unit of activity calculated to be approximately 1-2 ng. It is present in human serum, urine, and cerebrospinal fluid. The isolation procedure consists of initial aqueous and methanolic extractions followed by a series of HPLC chromatography steps (reverse phase and TSK sizing columns). The reverse-phase chromatography of CDS extracted under identical conditions from bovine brain and human serum show similar retention times. The final chromatography step gives a single active peak with a distinct ultraviolet spectrum, a single molecular peak m/z 587.8 +/- 2 in plasma desorption mass spectrometry (PDMS), and a unique pharmacological and physiological profile. Clonidine-displacing substance does not partition into organic solvents and it is ninhydrin and fluorescamine negative. All of these molecular properties clearly distinguish CDS from agmatine, an endogenous 130-dalton compound of far greater abundance which displays lower affinity for p-aminoclonidine-labeled sites in rat brain membranes. The ultraviolet spectrum of CDS consists of two aromatic peaks at 224 and 276 nm, whereas agmatine is an aliphatic substance with no ultraviolet absorbance. Like many antihypertensive drugs of the guanidine and imidazoline family of compounds, CDS recognizes alpha 2-adrenergic receptors, clonidine sites (IR-I1), and imidazoline sites (IR-I2). A good correlation exists between the affinities of various imidazoline/guanidine type ligands for IR-I2 in both human placenta and rat liver membranes which can be accurately determined because both tissues lack IR-I1 and alpha 2-adrenergic receptors. There is no correlation in the affinities of these ligands for IR-I2 of human-placental versus alpha 2-adrenergic receptors of human platelets. By uncovering the role of CDS in the central nervous system we will be able to understand the coupling of IRs to neurotransmission and, in turn, to changes in arterial pressure.