Scott C. Harvey

Differential Inhibition by α‐Conotoxin‐MII of the Nicotinic Stimulation of [3H]Dopamine Release from Rat Striatal Synaptosomes and Slices

Abstract: The presynaptic nicotinic modulation of dopamine release from striatal nerve terminals is well established, but the subtype(s) of neuronal nicotinic acetylcholine receptor (nAChR) underlying this response has not been identified. Recently, α‐conotoxin‐MII has been reported to inhibit potently and selectively the rat α3/β2 combination of nAChR subunits. Here we have synthesised the peptide, confirmed its specificity, and examined its effect on the (±)‐anatoxin‐a‐evoked release of [3H]dopamine from rat striatal synaptosomes and slices. α‐Conotoxin‐MII (112 nM) completely blocked acetylcholine‐evoked currents of α3β2 nAChRs expressed in Xenopus oocytes (IC50 = 8.0 ± 1.1 nM). Pairwise combinations of other nicotinic subunits were not blocked by 112 nMα‐conotoxin‐MII. On perfused striatal synaptosomes and slices, α‐conotoxin‐MII dose‐dependently inhibited [3H]dopamine release evoked by 1 µM (±)‐anatoxin‐a with IC50 values of 24.3 ± 2.9 and 17.3 ± 0.1 nM, respectively. The dose‐response curve was shifted to the right with increasing agonist concentrations. However, the maximal inhibition of responses achieved by α‐conotoxin‐MII (112 nM) was 44.9 ± 5.4% for synaptosomes and 25.0 ± 4.1% for slices, compared with an inhibition by 10 µM mecamylamine of 77.9 ± 3.7 and 88.0 ± 2.1%, respectively. These results suggest the presence of presynaptic α3β2‐like nAChRs on striatal dopaminergic terminals, but the incomplete block of (±)‐anatoxin‐a‐evoked [3H]dopamine release by α‐conotoxin‐MII also supports the participation of nAChRs composed of other subunits. The lower inhibition found in slices is consistent with an additional indirect nicotinic stimulation of dopamine release via an α‐conotoxin‐MII‐insensitive nAChR.

Multiple Determinants of Dihydro-β-Erythroidine Sensitivity on Rat Neuronal Nicotinic Receptor α Subunits

Abstract: Neuronal nicotinic acetylcholine receptors are differentially sensitive to blockade by the competitive antagonist dihydro‐β‐erythroidine. Both α and β subunits participate in determining sensitivity to this antagonist. The α subunit contribution to dihydro‐β‐erythroidine sensitivity is illustrated by comparing the α4β4 receptor and the α3β4 receptor, which differ in sensitivity to dihydro‐β‐erythroidine by ∼120‐fold. IC50 values for blocking α4β4 and α3β4, responding to EC20 concentrations of acetylcholine, were 0.19 ± 0.06 and 23.1 ± 10.2 µM, respectively. To map the sequence segments responsible for this difference, we constructed a series of chimeric α subunits containing portions of the α4 and α3 subunits. These chimeras were coexpressed with β4, allowing pharmacological characterization. We found determinants of dihydro‐β‐erythroidine sensitivity to be distributed throughout the N‐terminal extracellular domain of the α subunit. These determinants were localized to sequence segments 1–94, 94–152, and 195–215. Loss of determinants within segment 1–94 had the largest effect, decreasing dihydro‐β‐erythroidine sensitivity by 4.3‐fold.

Pharmacological and Behavioral Profile of N-(4-Fluorophenylmethyl)-N-(1-methylpiperidin-4-yl)-N′-(4-(2-methylpropyloxy)phenylmethyl) Carbamide (2R,3R)-Dihydroxybutanedioate (2:1) (ACP-103), a Novel 5-Hydroxytryptamine2A Receptor Inverse Agonist

The in vitro and in vivo pharmacological properties of N-(4-fluorophenylmethyl)-N-(1-methylpiperidin-4-yl)-N′-(4-(2-methylpropyloxy)phenylmethyl)carbamide (2R,3R)-dihydroxybutanedioate (2:1) (ACP-103) are presented. A potent 5-hydroxytryptamine (5-HT)2A receptor inverse agonist ACP-103 competitively antagonized the binding of [3H]ketanserin to heterologously expressed human 5-HT2A receptors with a mean pKi of 9.3 in membranes and 9.70 in whole cells. ACP-103 displayed potent inverse agonist activity in the cell-based functional assay receptor selection and amplification technology (R-SAT), with a mean pIC50 of 8.7. ACP-103 demonstrated lesser affinity (mean pKi of 8.80 in membranes and 8.00 in whole cells, as determined by radioligand binding) and potency as an inverse agonist (mean pIC50 7.1 in R-SAT) at human 5-HT2C receptors, and lacked affinity and functional activity at 5-HT2B receptors, dopamine D2 receptors, and other human monoaminergic receptors. Behaviorally, ACP-103 attenuated head-twitch behavior (3 mg/kg p.o.), and prepulse inhibition deficits (1-10 mg/kg s.c.) induced by the 5-HT2A receptor agonist (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride in rats and reduced the hyperactivity induced in mice by the N-methyl-d-aspartate receptor noncompetitive antagonist 5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate; MK-801) (0.1 and 0.3 mg/kg s.c.; 3 mg/kg p.o.), consistent with a 5-HT2A receptor mechanism of action in vivo and antipsychotic-like efficacy. ACP-103 demonstrated >42.6% oral bioavailability in rats. Thus, ACP-103 is a potent, efficacious, orally active 5-HT2A receptor inverse agonist with a behavioral pharmacological profile consistent with utility as an antipsychotic agent.