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

1. We characterized the binding of[H-3]-rauwolscine, [H-3]-p-aminoclon idine and [H-3]-idazoxan in a dog kidney membrane preparation. Our aim was to determine the pharmacological nature of the alpha(2)-adrenocep tor- and imidazoline-preferring binding sites in this organ. 2. [H-3]- 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, whe n 10 mu mol/L idazoxan defined non-specific binding. However displacem ent studies demonstrated that a number of compounds, including prazosi n, inhibited [H-3]-rauwolscine binding in a complex manner consistent with displacement from two distinct binding sites. The majority (69%) of the [H-3]-rauwolscine binding sites had a relatively low affinity f or prazosin (K-I = 398 nmol/L), while the remainder had a relatively h igh affinity for prazosin (K-I = 7.9 nmol/L). 3. [H-3]-p-Aminoclonidin e bound to an apparent single site (K-D = 5.2 nmol/L; B-max = 72.4 fmo l/mg protein), when 10 mu mol/L phentolamine defined non-specific bind ing. When 1 mu mol/L of the potent and selective alpha(2)-adrenoceptor antagonist 2-methoxyidazoxan was included in the incubate, no specifi c binding was detected. We therefore conclude that under the condition s of this experiment [H-3]-p-aminoclonidine binds only to alpha(2)-adr enoceptors in the dog kidney. 4. [H-3]-Idazoxan bound to two sites, wi th 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, respective ly, when 1 mmol/L phentolamine defined non-specific binding. When 10 m u mol/L GTP gamma S was included in the incubate, the low affinity sit e was unaffected but the maximum binding at the higher affinity site w as reduced by 79%. 2-Methoxyidazoxan displaced [H-3]-idazoxan in a mon ophasic manner and with low potency (IC50 = 11.5 mu mol/L). Yohimbine, efaroxan, clonidine, rilmenidine, guanabenz and idazoxan itself displ aced [H-3]-idazoxan in a complex manner; the slope of the displacement curves being less than unity. 5. We conclude that the dog kidney cont ains a heterogeneous population of alpha(2)-adrenoceptors that can be labelled either with [H-3]-rauwolscine or [H-3]-p-aminoclonidine. The dog kidney also contains a heterogeneous population of non-adrenocepto r imidazoline-preferring binding sites of the I-2-subtype, that can be labelled with [H-3]-idazoxan. The binding site for which [H-3]-idazox an has the highest affinity appears to be coupled to a guanine nucleot ide binding regulatory protein.