, Volume 348, Issue 1, pp 70-76

Binding of [3H]clonidine to I1-imidazoline sites in bovine adrenal medullary membranes

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Imidazolines bind with high affinity not only to α-adrenoceptors but also to specific imidazoline binding sites (IBS) labelled by either [3H]clonidine or [3H]idazoxan and termed I1- and I2-IBS, respectively. Since bovine adrenal chromaffin cells lack α2-adrenoceptors, we investigated the pharmacological characteristics of [3H]clonidine binding sites in the bovine adrenal medulla. The binding of [3H]clonidine was rapid, reversible, partly specific (as defined by naphazoline 0.1 mmol/l; 55% specific binding at [3H]clonidine 10 nmol/l), saturable and of high affinity. The specific binding of [3H]clonidine to bovine adrenal medullary membranes was concentration-dependently inhibited by various imidazolines, guanidines and an oxazoline derivative but not, or with negligible affinity, by rauwolscine and (−)-adrenaline. In most cases, the competition curves were best fitted to a two-site model. The rank order of affinity for the high affinity site (in a few cases the single detectable site) was as follows: naphazoline >- BDF 7579 (4-chloro-2-isoindolinyl guanidine) >-clonidine>- cirazoline >_ BDF 6143 (4-chloro-2-(2-imidazolin-2-ylamino)isoindoline hydrochloride) > BDF 7572 (4,7-chloro-2-(2-imidazolin-2-ylamino)-isoindoline) > moxonidine = rilmenidine > BDF 6100 (2-(2-imidazolin-2-ylamino)-isoindoline) = idazoxan > phentolamine > aganodine = guanabenz > amiloride > histamine. This rank order is compatible with the pharmacological properties of the I1-IBS. The non-hydrolysable GTP-analogue Gpp(NH)p (5′guanylylimidodiphosphate; 100 μmol/l) inhibited specific [3H]clonidine binding by about 50%. Equilibrium [3H]clonidine binding was also significantly reduced by K+ and Mg2+ In conclusion, [3H]clonidine labels non-adrenergic high-affinity sites in plasma membranes of the bovine adrenal medulla; these sites exhibit the pharmacological properties of I1-IBS, but not of I2-IBS. Furthermore, the IBS in the adrenal medulla appear to be coupled to a G-protein.

Correspondence to G. J. Molderings at the above address