Expression of non-adrenergic imidazoline sites in chromaffin cells and mitochondrial membranes of bovine adrenal medulla.

We sought to characterize the non-adrenergic binding site for imidazolines, the imidazoline receptor in whole membranes and subcellular compartments of chromaffin cells of bovine adrenal medulla. [3H]Idazoxan exhibited saturable and high affinity (KD = 5 nM) binding to chromaffin cell membranes fully displaceable by idazoxan and cirazoline but not by epinephrine or rauwolscine. Binding sites were highly enriched in mitochondrial but not plasma membranes and absent from nuclear fractions. The rank order of potency for displacement of [3H]idazoxan from mitochondrial membranes was: cirazoline > idazoxan > naphazoline > amiloride > detomedine > clonidine >> phentolamine > cimetidine = imidazole 4-acetic acid > p-iodoclonidine = epinephrine = norepinephrine = rauwolscine. Binding was also inhibited with high affinity by the purported endogenous ligand clonidine-displacing substance and by K+ and the K(+)-channel antagonists 4-aminopyridine and tetraethylammonium bromide but not Na+. We conclude that: (a) adrenal chromaffin cells express imidazoline receptors but not alpha 2-adrenergic receptors; (b) these sites are predominantly localized to adrenal medullary mitochondria; and (c) imidazoline receptors conform to an idazoxan preferring (I-2) rather than the clonidine preferring (I-1) subclass and are amiloride sensitive. The data support the view that alpha 2-adrenergic and imidazoline receptors are distinct receptor species and that adrenal chromaffin cells would be a useful cultured cell system, expressing only imidazoline receptors, for further molecular and functional studies of the receptors.