William C. Randall

Multiple central WB-4101 binding sites and the selectivity of prazosin

Under the conditions employed in a standard displacement assay of [3H]WB-4101 from calf neocortical membrane homogenates (0.22 nM total [3H]WB-4101), 70–80% of this radioligand is bound to a high affinity receptor site and 20–30% to a second, lower affinity site. This latter site is not the one previously shown to bind alpha agonists. Binding parameters for WB-4101 to each site have been determined. Prazonin is extremely selective for the high affinity site as indicated by a biphasic displacement of [3H]WB-4101.

Pharmacological profile of a new potent and specific α2-adrenoceptor antagonist, L-657,743

SummaryL-657,743, (2S,12bS)1′,3′-dimethylspiro (1, 3,4,5′, 6,6′, 7,12 b-octahydro-2-H-benzo[b]furo[2,3-a]quinolizine)-2, 4′- pyrimidin-2′-one, was tested in several in vitro and in vivo models for α2-adrenoceptor antagonism. L-657,743 exhibited a high affinity (⩽ 1 nM) for α2-adrenoceptors labelled by [3H] rauwolscine or [3H]clonidine with a 240-fold selectivity versus α1-adrenoceptors labelled by [3H]prazosin. L-657,743 was a potent, selective, and competitive α2-adrenoceptor antagonist in the rat isolated vas deferens (pA2 = 9.3 vs clonidine; pA2 = 7.1 vs methoxamine). In vivo, L-657,743 potently blocked clonidine-induced mydriasis in the rat and stimulated cerebrocortical norepinephrine synthesis, two indices of central α2-adrenoceptor antagonism. L-657,743 exhibited a comparatively low affinity for several monoamine receptor subtypes (D1, D2, 5-HT1, 5-HT2) in radioligand binding assays in vitro and a comparatively low potency to alter the synthesis of brain DA and 5-HT in vivo indicating a marked α2-specificity versus other monoamine receptor mechanisms. Compared to yohimbine, L-657,743 had considerably higher α2-antagonist potency and α2/α1 selectivity and was significantly more α2-specific (i.e., vs. DA, 5-HT receptors).

Multiple central α2 adrenoceptors of avian and mammalian species

Abstract Although equilibrium binding experiments indicated that calf cerebral membranes contained two classes of clonidine receptors and that chicken cerebral membranes might have contained only one, experiments investigating the kinetics of binding and the effects of GppNHp clearly indicated that the cerebral membranes of both species contained two subtypes of receptor, with the avian high affinity receptor having been present at too low a density to be readily detected in equilibrium binding studies. For both species 10 μM GppNHp sharply reduced or eliminated both the high affinity binding site and the slow steps of association and dissociation without changing the low affinity site and its related rapid association and dissociation steps. The high affinity sites from both species had similar specificities since the relative affinities of the avian binding site for a series of clonidine analogues closely reflected the relative affinities of the calf binding site. The properties of the chicken and calf α 2 subtypes resembled those reported for rat brain.

L-654,284 a new potent and selective α2-adrenoceptor antagonist

SummaryL-654,284 ((2R, 12bS)-N-(1,3,4,6,7,12b-hexahydro-2H-benzo[b]-furo[2,3-a] quinolizine-2-yl)-N-methyl-2-hydroxyethanesulfonamide) was tested in several in vitro and in vivo models for α2-adrenoceptor antagonist activity and compared to several reference agents. In vitro L-654,284 competed for the binding of 3H-clonidine or 3H-rauwolscine (Kis 0.8 nM, 1.1 nM) and blocked the presynaptic effects of clonidine in the rat isolated vas deferens (pA2, 9.1). L-654,284 exhibited marked α2- vs. α1-adrenoceptor selectivity in vitro, inhibiting 3H-prazosin binding with a Ki of 110 nM and blocking the effects of methoxamine on the vas deferens with a pA2 of 7.5. In vivo L-654,284 at 22 nmoles/kg i.v. doubled the ED50 of clonidine to produce mydriasis in rats. Given orally, the potency of L-654,284 in this test was reduced by a factor of 5.5. L-654,284 also potently increased cerebrocortical NE turnover in the rat, another in vivo index of α2-adrenoceptor blockade in the central nervous system. In the periphery, L-654,284 demonstrated α2-adrenoceptor selectivity by preferentially blocking the pressor effects of UK 14304 versus those of methoxamine in the pithed rat. Overall, L-654,284 was generally a more potent α2-adrenoceptor antagonist than RX 781094 with comparable α2/α1 selectivity and was several times more potent and α2-selective than WY 26703 or yohimbine. In addition, L-654,284 had better (5–6 times) oral bioavailability than RX 781094 or WY 26703.

[3H]L-654,284 as a probe of the central alpha2 adrenoceptor

SummaryL-654,284 ((2R, 12bS)-N-(1,3,4,6,7,12b-hexahydro-2H-benzo[b]-furo[2,3-a]quinolizin-2-yl)-N-methyl-2-hydroxyethanesulfonamide), a potent and selective antagonist of the alpha2 adrenoceptor, was tritiated to high specific activity. Saturation binding to cell membrane suspensions obtained from calf cerebral cortex revealed a high affinity binding site (0.63 nM). Kinetics of association and dissociation were well represented by single exponential processes, and the equilibrium dissociation constant obtained from the ratio of rate constants agreed well with that found by saturation binding. A direct comparison of saturation binding revealed that the antagonist [3H]L-654,284 had roughly the same affinity for the alpha2 adrenoceptor as the agonist [3H]clonidine and eight times the affinity of the antagonist [3H]rauwolseine. The maximum receptor densities of these radioligands were not significantly different. Competition assays with a series of compounds of known receptor affinity revealed that [3H]L-654,284 selectively binds to a site with all of the characteristics expected of the alpha2 adrenoceptor.