CHARACTERIZATION OF BINDING SITES FOR [³H]‐IDAZOXAN, [³H]‐P‐AMINOCLONIDINE AND [³H]‐RAUWOLSCINE IN THE KIDNEY OF THE DOG

1. We characterized the binding of [³H]‐rauwolscine, [³H]‐p‐aminoclonidine and [³H]‐idazoxan in a dog kidney membrane preparation. Our aim was to determine the pharmacological nature of the α₂‐adrenoceptor‐ and imidazoline‐preferring binding sites in this organ. 2. [³H]‐Rauwolscine bound to an apparent single site with an affinity (K_D) of 2.2 nmol/ L and a maximum density (B_max) of 58.5 fmol/mg protein, when 10 μmol/L idazoxan defined non‐specific binding. However displacement studies demonstrated that a number of compounds, including prazosin, inhibited [³H]‐rauwolscine binding in a complex manner consistent with displacement from two distinct binding sites. The majority (69%) of the [³H]‐rauwolscine binding sites had a relatively low affinity for prazosin (K_I= 398 nmol/L), while the remainder had a relatively high affinity for prazosin (K_I= 7.9 nmol/ L). 3. [³H]‐p‐Aminoclonidine bound to an apparent single site (K_D= 5.2 nmol/L; B_max= 72.4 fmol/mg protein), when 10 μmol/L phentolamine defined non‐specific binding. When 1 μmol/L of the potent and selective α₂‐adrenoceptor antagonist 2‐methoxyidazoxan was included in the incubate, no specific binding was detected. We therefore conclude that under the conditions of this experiment [³H]‐p‐aminoclonidine binds only to α₂‐adrenoceptors in the dog kidney. 4. [³H]‐Idazoxan bound to two sites, with a higher (K_D= 0.95 nmol/L; B_max= 43.9 fmol/mg protein) and lower (K_D= 9.1 nmol/L; B_max= 93.8 fmol/mg protein) affinity, respectively, when 1 mmol/L phentolamine defined non‐specific binding. When 10 μmol/ L GTPγS was included in the incubate, the low affinity site was unaffected but the maximum binding at the higher affinity site was reduced by 79%. 2‐Methoxyidazoxan displaced [³H]‐idazoxan in a monophasic manner and with low potency (IG₅₀=11.5 μmol/L). Yohimbine, efaroxan, clonidine, rilmenidine, guanabenz and idazoxan itself displaced [³H]‐idazoxan in a complex manner; the slope of the displacement curves being less than unity. 5. We conclude that the dog kidney contains a heterogeneous population of α₂‐adrenoceptors that can be labelled either with [³H]‐rauwolscine or [³H]‐p‐aminoclonidine. The dog kidney also contains a heterogeneous population of non‐adrenoceptor imidazoline‐preferring binding sites of the I₂‐subtype, that can be labelled with [³H]‐idazoxan. The binding site for which [³H]‐idazoxan has the highest affinity appears to be coupled to a guanine nucleotide binding regulatory protein.