[3H]idazoxan binding to bovine adrenal medullary membranes: identification and pharmacological characterization of I2-imidazoline sites.

Bovine adrenal chromaffin cells, which have been shown to lack alpha 2-adrenoceptors, were used to investigate the pharmacological characteristics of [3H]idazoxan binding sites. The binding of [3H]idazoxan was very rapid, reversible, partly specific (as defined by cirazoline 0.1 mmol/l; 50% specific binding at [3H]idazoxan 10 nmol/l), saturable and of high affinity (KD 13nmol/l) and, hence, was compatible with the criteria for the identification of an imidazoline binding site (IBS). Since in competition experiments rauwolscine and (-)-adrenaline showed only negligible affinity for these adrenal medullary binding sites, the lack of alpha 2-adrenoceptors was confirmed. Histamine and amiloride also did not inhibit [3H]idazoxan binding or caused only negligible inhibition. In contrast, the specific binding of [3H]idazoxan was concentration-dependently inhibited by several imidazolines and guanidines with the following rank order of potency which conforms to the characteristics of the previously defined I2-IBS (in parentheses: Ki, 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-yl-amino)-isoindoline) > or = BDF 7579 (868; (4-chloro-2-isoindolinyl)guanidine) > phentolamine (1424) = naphazoline (1451). Equilibrium [3H]idazoxan binding was reduced by K+ but not by Na+ or the non-hydrolysable GTP-analogue Gpp(NH)p (5'-guanylylimidodiphosphate; 100 mumol/l). In conclusion, membranes of the bovine adrenal medulla are endowed with non-adrenergic high-affinity [3H]idazoxan sites which exhibit the pharmacological properties of the amiloride-insensitive subtype of I2-IBS and probably are not coupled to a G-protein.