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 (KD) of 2.2 nmol/ L and a maximum density (Bmax) 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 (KI= 398 nmol/L), while the remainder had a relatively high affinity for prazosin (KI= 7.9 nmol/ L). 3. [3H]‐p‐Aminoclonidine bound to an apparent single site (KD= 5.2 nmol/L; Bmax= 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 (KD= 0.95 nmol/L; Bmax= 43.9 fmol/mg protein) and lower (KD= 9.1 nmol/L; Bmax= 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.
|Number of pages||10|
|Journal||Clinical and Experimental Pharmacology and Physiology|
|Publication status||Published - 1994|
- imidazoline‐preferring receptor
- radioligand binding