[H-3] IDAZOXAN BINDING TO BOVINE ADRENAL-MEDULLARY MEMBRANES - IDENTIFICATION AND PHARMACOLOGICAL CHARACTERIZATION OF I-2-IMIDAZOLINE SITES

Bovine adrenal chromaffin cells, which have been shown to lack alpha(2 )-adrenoceptors, were used to investigate the pharmacological characte ristics of [H-3]idazoxan binding sites. The binding of [H-3]idazoxan w as very rapid, reversible, partly specific (as defined by cirazoline 0 .1 mmol/l; 50% specific binding at [H-3]idazoxan 10 nmol/l), saturable and of high affinity (K-D 13 nmol/l) and, hence, was compatible with the criteria for the identification of an imidazoline binding site (IB S). Since in competition experiments rauwolscine and (-)-adrenaline sh owed only negligible affinity for these adrenal medullary binding site s, the lack of alpha(2)-adrenoceptors was confirmed. Histamine and ami loride also did not inhibit [H-3]idazoxan binding or caused only negli gible inhibiton. In contrast, the specific binding of [H-3]idazoxan wa s concentration-dependently inhibited by several imidazolines and guan idines with the following rank order of potency which conforms to the characteristics of the previously defined I-2-IBS (in parentheses: K-i , nmol/l): idazoxan (4) = cirazoline (4) >> clonidine (272) = BDF 6143 (299; 4-chloro-2-(2-imidazoline-2-ylamino)isoindoline)> BDF 6100 (563 ; 2-(2-imidazolin-2-ylamino)-isoindoline) greater than or equal to BDF 7579 (868; (4-chloro-2-isoindolinyl)guanidine) > phentolamine (1424) = naphazoline (1451). Equilibrium [H-3]idazoxan binding was reduced by K+ but not by Naf or the non-hydrolysable GTP-analogue Gpp(NH)p (5'-g uanylylimidodiphosphate; 100 mu mol/l). In conclusion, membranes of th e bovine adrenal medulla are endowed with non-adrenergic high-affinity [H-3]idazoxan sites which exhibit the pharmacological properties of t he amiloride-insensitive subtype of I-2-IBS and probably are not coupl ed to a G-protein.