Identification of ?(2) adrenergic receptors in human frontal cortex membranes by binding of [(3)H]RX 821002, the 2-methoxy analog of [(3)H]idazoxan.

The antagonist [(3)H]idazoxan binds with comparable affinity to ?(2) adrenergic receptors and to phentolamine-displaceable non-stereoselective sites in human frontal cortex membranes. In contrast, idazoxan analogs possessing alkyl and alkoxy substituents at the 2-position of the benzodioxan moiety (i.e. RX 821002: 2-methoxy-1,4-[6,7-(3)H]benzodioxan-2-yl-2-imidazolin HCl, 43.8 Ci/mmol) possess 300-1200 times lower affinity for the non-stereoselective sites. Their affinity for the ?(2) receptors is increased as well, resulting in more than a 1000-fold selectivity towards the receptors as compared to the non-stereoselective sites. [(3)H]RX 821002, the 2-methoxy analog of idazoxan possesses an approx. 10-fold higher affinity for the ?(2) receptors (K(D) = 2.8 nM than [(3)H]idazoxan (K(D) = 24 nM) and about equal affinity as [(3)H]rauwolscine (K(D) = 3.6 nM). [(3)H]Rauwolscine binds with comparable affinity to ?(2) receptors and to 5-HT(1A) receptors, and competition studies indicate that the K(i) value of unlabelled RX 821002 for the 5-HT(1A) receptors (30 nM) is about one order in magnitude above its K(i) value for the ?(2) receptors (4.1 nM). Labelling of the 5-HT(1A) receptors by [(3)H]RX 821002 and by [(3)H]rauwolscine can be prevented by selective masking with 8-OH-DPAT (30 nM) or 5-HT (0.3 ?M). Under these conditions, specific binding of [(3)H]RX 821002 to the ?(2) receptors represents 84% of total binding (at its K(D)), as compared to 77% for [(3)H]rauwolscine and 20% for [(3)H]idazoxan. [(3)H]RX 821002 labels the ?(2) receptors as a single class of non-cooperative sites. Association and dissociation kinetics are very fast at 37 degrees C. Antagonist competition curves are steep with Hill coefficients close to one and the agonist curves can be analysed in terms of two affinity sites, confirming the antagonistic properties of [(3)H]RX821002. About 60% of the ?(2) receptors possess high agonist affinity.