Epibatidine, a nicotinic acetylcholine receptor agonist, inhibits the capsaicin response in dorsal root ganglion neurons.

In patch-clamp recordings from small-medium diameter dorsal root ganglion neurons in culture, (+/-)-epibatidine (1 microM) was able to inhibit the capsaicin response (IC(50)=0.32 microM) in neurons where there was no detectable direct nicotinic response. Thus, (+/-)-epibatidine may inhibit the vanilloid receptor in a manner that is not dependent upon nicotinic current activation, representing another mechanism by which such ligands could modulate vanilloid receptor signaling.

Gain of function mutation of the alpha7 nicotinic receptor: distinct pharmacology of the human alpha7V274T variant.

In the human alpha7 nicotinic receptor, valine-274 in the pore-lining transmembrane-2 region was mutated to threonine to produce the variant human alpha7V274T, which was evaluated electrophysiologically following expression in Xenopus laevis oocytes. Inward current rectification was strong in human alpha7V274T as in the human alpha7 wild type nicotinic receptor. However, human alpha7V274T was 100-fold more sensitive to the nicotinic receptor agonists acetylcholine, (-)-nicotine and 1,1-dimethyl-4-phenylpiperazinium. Choline also activated human alpha7V274T (EC50 = 12 microM) and was 82-fold more potent than at human alpha7 wild type nicotinic receptor. (-)-Cotinine, (2,4)-dimethoxybenzylidene anabaseine (GTS-21) and 2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine (ABT-089), weak partial agonists at human alpha7 wild type, were much stronger agonists at human alpha7V274T with EC50 values of 70 microM, 4 microM and 28 microM and fractional activation values of 93%, 96% and 40%, respectively. However, (-)-lobeline, a human alpha7 wild type nicotinic receptor antagonist, and dihydro-beta-erythroidine, which activates chick mutagenized alpha7 nicotinic receptors, had only weak agonist-like activity at human alpha7V274T (< or = 4% of the maximal acetylcholine response). Methyllycaconitine, mecamylamine, d-tubocurarine and dihydro-beta-erythroidine retained antagonist activity and, indeed, appeared to be at least as potent at human alpha7V274T as at human alpha7 wild type. These results support and extend the concept that human nicotinic receptor pharmacology can be profoundly altered by single amino acid changes in the pore-lining segment.

Activation and inhibition of the human alpha7 nicotinic acetylcholine receptor by agonists.

To better understand the effects of weak as well as strong agonists at the human alpha7 nicotinic acetylcholine receptor (human alpha7 nAChR), the abilities of several classic nAChR agonists to both activate and inhibit (desensitize) the human alpha7 nAChR expressed in Xenopus oocytes were quantified and compared. Activation was measured during 0.2-20 s agonist application, as required to elicit a peak response. Inhibition was measured as the reduction in the agonist response to 200 microM ACh in the presence of inhibitor during a 5-20 min incubation. Acetylcholine (ACh), (-)-nicotine, (+)-nicotine, and 1,1-dimethyl-4-phenylpiperazinium (DMPP) were 62- to 130-fold more potent as inhibitors than as activators, with excellent correlation between the IC50 and EC50 values (r2 = 0.924). Agonist concentrations that elicited only 0.6-1.2% nAChR activation were sufficient to inhibit the response to ACh by 50%. Thus, even a very weak agonist could appear to be a potent and effective inhibitor through receptor desensitization. (-)-Lobeline, in contrast, acted as an antagonist at the human alpha7 nAChR, eliciting no detectable agonist-like response at concentrations up to 1 mM, but inhibiting the response to ACh with an IC50 value of 8.5 microM. (-)-Cotinine and the novel ligand ABT-089 [2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine] acted as weak agonists at the human alpha7 nAChR (1 and 1.5% response at 1 mM, respectively) and inhibited the response to ACh with IC50) values of 175 and 48 microM, respectively. These effects could be explained by receptor desensitization, at least in part.

ABT-594 [(R)-5-(2-azetidinylmethoxy)-2-chloropyridine]: a novel, orally effective analgesic acting via neuronal nicotinic acetylcholine receptors: I. In vitro characterization.

The discovery of (+/-)-epibatidine, a naturally occurring neuronal nicotinic acetylcholine receptor (nAChR) agonist with antinociceptive activity 200-fold more potent than that of morphine, has renewed interest in the potential role of nAChRs in pain processing. However, (+/-)-epibatidine has significant side-effect liabilities associated with potent activity at the ganglionic and neuromuscular junction nAChR subtypes which limit its potential as a clinical entity. ABT-594 [(R)-5-(2-azetidinylmethoxy)-2-chloropyridine] is a novel, potent cholinergic nAChR ligand with analgesic properties (see accompanying paper by Bannon et al., 1998b) that shows preferential selectivity for neuronal nAChRs and a consequently improved in vivo side-effect profile compared with (+/-)-epibatidine. ABT-594 is a potent inhibitor of the binding of [3H](-)-cytisine to alpha 4 beta 2 neuronal nAChRs (Ki = 37 pM, rat brain; Ki = 55 pM, transfected human receptor). At the alpha 1 beta 1 delta gamma neuromuscular nAChR labeled by [125I] alpha-bungarotoxin (alpha-Btx), ABT-594 has a Ki value of 10,000 nM resulting in a greater than 180,000-fold selectivity of the compound for the neuronal alpha 4 beta 2 nAChR. In contrast, (+/-)-epibatidine has Ki values of 70 pM and 2.7 nM at the alpha 4 beta 2 and alpha 1 beta 1 delta gamma nAChRs, respectively, giving a selectivity of only 38-fold. The S-enantiomer of ABT-594, A-98593 has activity at the neuronal alpha 4 beta 2 nAChR identical with ABT-594 (Ki = 34-39 pM), which demonstrates a lack of stereospecific binding similar to that reported previously for (+/-)-epibatidine. A similar lack of stereoselectivity is seen at the human alpha 7 receptor. However, A-98593 is 3-fold more potent at the neuromuscular nAChR (Ki = 3420 nM) and the brain alpha-Btx-sensitive nAChR (Ki = 4620 nM) than ABT-594. ABT-594 has weak affinity in binding assays for adrenoreceptor subtypes alpha-1B (Ki = 890 nM), alpha-2B (Ki = 597 nM) and alpha-2C (Ki = 342 nM), and it has negligible affinity (Ki > 1000 nM) for approximately 70 other receptors, enzyme and transporter binding sites. Functionally, ABT-594 is an agonist. At the transfected human alpha 4 beta 2 neuronal nAChR (K177 cells), with increased 86Rb+ efflux as a measure of cation efflux, ABT-594 had an EC50 value of 140 nM with an intrinsic activity (IA) compared with (-)-nicotine of 130%; at the nAChR subtype expressed in IMR-32 cells (sympathetic ganglion-like), an EC50 of 340 nM (IA = 126%); at the F11 dorsal root ganglion cell line (sensory ganglion-like), an EC50 of 1220 nM (IA = 71%); and via direct measurement of ion currents, an EC50 value of 56,000 nM (IA = 83%) at the human alpha 7 homooligimeric nAChR produced in oocytes. A-98593 is 2- to 3-fold more potent and displays approximately 50% greater intrinsic activity than ABT-594 in all four functional assays. In terms of potency, ABT-594 is 8- to 64-fold less active than (+/-)-epibatidine and also has less IA in these functional assays. ABT-594 (30 microM) inhibits the release of calcitonin gene-related peptide from C-fibers terminating in the dorsal horn of the spinal cord, an effect mediated via nAChRs. Pharmacologically, ABT-594 has an in vitro profile distinct from that of the prototypic nicotinic analgesic (+/-)-epibatidine, with the potential for substantially reduced side-effect liability and, as such, represents a potentially novel therapeutic approach to pain management.

ABT-089 [2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine]: I. A potent and selective cholinergic channel modulator with neuroprotective properties.

Accumulating preclinical and clinical evidence data suggests that compounds that selectively activate neuronal nicotinic acetylcholine receptor (nAChR) subtypes may have therapeutic utility for the treatment of several neurological disorders. In the present study, the in vitro pharmacological properties of the novel cholinergic channel modulator ABT-089 [2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine], are described. In radioligand binding studies, ABT-089 was shown to display selectivity toward the high-affinity (-)-cytisine binding site present on the alpha4beta2 nAChR subtype (Ki = 16 nM) relative to the [125I]alpha-bungarotoxin binding site present on the alpha7 (Ki > or = 10,000 nM) and alpha1beta1deltagamma (Ki > 1000 nM) nAChR subtypes. In cation flux and channel current studies, ABT-089 displayed a more complex profile than (-)-nicotine having agonist, partial agonist and inhibitory activities depending on the nAChR subtype with which it interacts. ABT-089 differentially stimulated neurotransmitter release. The compound displayed a similar potency and efficacy to (-)-nicotine to facilitate ACh release (ABT-089, EC50 = 3 microM; (-)-nicotine, EC50 = 1 microM), but was markedly less potent and less efficacious than (-)-nicotine to stimulate dopamine release (ABT-089, EC50 = 1.1 microM; (-)-nicotine, EC50 = 0.04 microM). Additionally, ABT-089 was neuroprotective against the excitotoxic insults elicited by exposure to glutamate in both rat cortical cell cultures (EC50 = 10 +/- 3 microM) and differentiated human IMR32 cells (EC50 = 3 +/- 2 microM). The differential full agonist/partial agonist profile of ABT-089, as compared with (-)-nicotine and ABT-418, illustrates the complexity of nAChR activation and the potential to target responses at subclasses of the neuronal and peripheral receptors.

A-85380 [3-(2(S)-azetidinylmethoxy) pyridine]: in vitro pharmacological properties of a novel, high affinity alpha 4 beta 2 nicotinic acetylcholine receptor ligand.

The in vitro pharmacological properties of a novel cholinergic channel ligand, A-85380 [3-(2(S)-azetidinylmethoxy)pyridine], were examined using tissue preparations that express different putative nAChR subtypes. In radioligand binding studies, A-85380 is shown to be a potent and selective ligand for the human alpha 4 beta 2 nAChR subtype (Ki = 0.05 + 0.01 nM) relative to the human alpha 7 (Ki = 148 +/- 13 nM) and the muscle alpha 1 beta 1 dg subtype expressed in Torpedo electroplax (Ki = 314 +/- 12 nM). The R-enantiomer of A-85380, A-159470, displays little enantioselectivity towards the alpha 4 beta 2 and alpha 1 beta 1 delta gamma subtypes but does not display 12-fold enantioselectivity towards the alpha 7 subtype (Ki = 1275 +/- 199 nM). (+)- and(-)-Epibatidine display similar potencies at the human human alpha 4 beta 2 (Ki = 0.04 +/- 0.02 nM and 0.07 +/- 0.02 nM, respectively), human alpha 7 (Ki = 16 +/- 2 nM and 22 +/- 3 nM, respectively) and muscle alpha 1 beta 1 delta gamma g (Ki = 2.5 +/- 0.9 nM and 5.7 +/- 1.0 nM, respectively) nAChRs. Functionally, A-85380 is a potent activator of cation efflux through the human alpha 4 beta 2 (EC50 = 0.7 +/- 0.1 microM) and ganglionic (EC50 = 0.8 +/- 0.09 microM) subtypes, effects that are attenuated by pretreatment with mecamylamine (10 microM). Further, A-85380 can activate (EC50 = 8.9 +/- 1.9 microM) currents through channels formed by injection of the human alpha 7 subunit into Xenopus oocytes, effects that are attenuated by pretreatment with the alpha 7 nAChR antagonist, methyllycaconitine (10 nM). In all cases, A-85380 is more potent than (-)-nicotine but less potent than (+/-)-epibatidine. In neurotransmitter release studies, A-85380 stimulates the release of dopamine with an EC 50 value of 0.003 +/- 0.001 microM which is equipotent to (+/-)-epibatidine, and 20-fold more potent than (-)-nicotine (EC50 = 0.04 +/- 0.009 microM). Thus, A-85380 displays a profile of robust activation of a number of nAChR subtypes with substantially less affinity for [125I] alpha-BgT sites than [3H](-)-cytisine sites, suggesting that it may serve as a more selective pharmacologic probe for the alpha 4 beta 2 subtype relative to the alpha 7 and alpha 1 beta 1 delta g nAChRs than (+/-)-epibatidine.

Effect of MK-801 at the human alpha 7 nicotinic acetylcholine receptor.

Responses of the human alpha 7 nicotinic acetylcholine receptor (alpha 7 nAChR) expressed in Xenopus laevis oocytes were quantified in the presence of barium (10 mM) to prevent secondary activation of Ca(2+)-dependent Cl- currents and atropine (2 microM) to block endogenous muscarinic receptors. Acetylcholine (ACh) elicited responses with EC50 values of 177 +/- 32 microM to 272 +/- 26 microM in different experiments. Responses to ACh (200 microM) were blocked by the nAChR antagonists alpha-bungarotoxin (IC50 = 0.54 +/- 0.04 nM), methyllycaconitine (IC50 = 0.64 +/- 0.08 nM) and mecamylamine (IC50 = 1.8 +/- 02 microM). Additionally, MK-801, a non-competitive blocker of N-methyl-D-aspartate (NMDA) sensitive glutamate receptor channels, inhibited the human alpha 7 nAChR. This effect was not stereoselective; the IC50 for (+)-MK-801 was 15 +/- 3 microM while that for (-)-MK-801 was 14 +/- 3 microM. The inhibition by MK-801, in contrast to methyllycaconitine, was dependent upon cell potential, consistent with a mechanism involving channel blockade. The inhibition by MK-801 reversed slowly (time constant approximately 20 min) compared to that by methyllycaconitine (100% recovery within 10 min). However, MK-801 did not appear to be trapped in the channel because the recovery from inhibition showed little dependence upon stimulation rate or cell potential. Thus, MK-801 acted as a non-stereoselective alpha 7 nAChR inhibitor that was only about 8-fold less potent than the nAChR antagonist mecamylamine and probably acted through channel blockade.

Human alpha 7 nicotinic acetylcholine receptor responses to novel ligands.

Responses of the human alpha 7 nicotinic acetylcholine receptor (nAChR) in Xenopus laevis oocytes were quantified using two-electrode voltage clamp in the presence of barium (10 mM) to block secondary activation of Ca(2+)-dependent chloride currents. The effect of (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl) isoxazole (ABT-418) and (2,4)-dimethoxybenzylidene anabaseine (GTS-21), two potential compounds for the treatment of Alzheimer's Disease, and of the natural product (+/-)epibatidine were compared to (-)nicotine. (+/-)Epibatidine acted as an agonist and was 64-fold more potent than (-) nicotine (EC50s = 1.30 +/- 0.11 microM and 83 +/- 10 microM, respectively). ABT-418 also was an agonist, 3-fold less potent and 75% as efficacious as (-)nicotine (EC50 = 264 +/- 34 microM). GTS-21, in contrast, inhibited the response to (-)nicotine at concentrations < or = 10 microM and itself elicited only a small response at higher concentrations (12% of the (-)nicotine response at 1 mM). Reversible blockade by methyllycaconitine (10 nM) corroborated the responses as due to activation of alpha 7 nAChR. This represents the first characterization of human alpha 7 nAChR responses to these novel nicotinic agonists.

(+-)-N6-endonorbornan-2-yl-9-methyladenine (N-0861) and its enantiomers: selective antagonists of A1-adenosine receptors in guinea pig isolated atria.

Adenosine and its metabolically stable analog 5'-N-ethylcarbox-amidoadenosine (NECA) induce negative inotropic, chronotropic and dromotrpic actions in the heart through activation of A1-adenosine receptors and relaxation of vascular smooth muscle through activation of A2-adenosine receptors. In vitro studies were carried out in order to determine the potency of the antagonist (+-)-N6-endonorbornan-2-yl-9-methyladenine (N-0861) and its two component enantiomers, WRC-0006(+) and WRC-0007(-), at the A1 receptors in the guinea pig atria and the A2 receptors in the guinea pig aorta. N-0861 competitively antagonized the negative inotropic responses induced by NECA in the eletrically paced left atrium (pKB = 6.24) and the negative chronotropic responses induced by NECA in the spontaneously beating right atrium (pKB = 6.29). WRC-0007 was 4-fold more potent (pKB = 6.51) than WRC-0006 (pKB = 5.86) at antagonizing the A1-adenosine receptors in the guinea pig left atrium. N-0861, WRC-0007 and WRC-0006 at high concentrations (> 3 x 10(-5) M) produced direct relaxations of the guinea pig aorta that masked to a small extent the A2 receptor antagonism by these compounds. The affinities of the antagonists for the A2 receptor in the aorta were calculated using the method of pharmacological resultant analysis. N-0861 was 47-fold less potent at the A2 receptor (pKB = 4.57) than it was at the A1 receptor. WRC-0006 was 2-fold more potent (pKB = 4.81) than WRC-0007 (pKB = 4.52) at the A2-adenosine receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-related changes in the synaptic plasticity of rat superior cervical ganglia.

A few seconds of tetanic preganglionic stimulation of rat sympathetic ganglia results in potentiation of cholinergic synaptic transmission lasting several hours. However, ganglia from aged (28-32 months) rats did not develop as much potentiation as did ganglia from young (3-6 months) rats. This potentiation appears to decay exponentially but in two phases. The early component decays in 15 minutes, a time course consistent with the phenomenon of post-tetanic potentiation (PTP). The later component decays over several hours and is a form of long-term potentiation (LTP). A double exponential decay model was employed to quantitatively resolve the decay of potentiation and allow quantitative comparison of the decay parameters in both aged and young rats. It is clear that the magnitude and duration of PTP was the same in both age groups. However both the magnitude and decay time-constant for LTP were 30-50% smaller in the aged group of rats. Several agents which mimic cyclic AMP or stimulate cyclic AMP production in the ganglion enhance nicotinic transmission for several hours. However, these agents, 8-bromo cAMP, forskolin, isoproterenol, and secretin were equally effective in potentiating transmission in ganglia from both young and aged rats. Previous studies demonstrated that stimulus induced LTP and cyclic AMP induced potentiation was the result of enhanced release of acetylcholine (ACh) and not from increased postsynaptic responsiveness. Presumably these agents act through the same pathway and by the same mechanism that generates LTP following preganglionic tetany. These observations demonstrate that there is a selective age-dependent decline in the capacity for sympathetic ganglia to generate long-term changes in synaptic efficacy.(ABSTRACT TRUNCATED AT 250 WORDS)