Effects of quinine, quinidine, and chloroquine on alpha9alpha10 nicotinic cholinergic receptors

In this study, we report the effects of the quinoline derivatives quinine, its optical isomer quinidine, and chloroquine on alpha9alpha10-containing nicotinic acetylcholine receptors (nAChRs). The compounds blocked acetylcholine (ACh)-evoked responses in alpha9alpha10-injected Xenopus laevis oocytes in a concentration-dependent manner, with a rank order of potency of chloroquine (IC50 = 0.39 microM) > quinine (IC50 = 0.97 microM) approximately quinidine (IC50= 1.37 microM). Moreover, chloroquine blocked ACh-evoked responses on rat cochlear inner hair cells with an IC50 value of 0.13 microM, which is within the same range as that observed for recombinant receptors. Block by chloroquine was purely competitive, whereas quinine inhibited ACh currents in a mixed competitive and noncompetitive manner. The competitive nature of the blockage produced by the three compounds was confirmed by equilibrium binding experiments using [3H]methyllycaconitine. Binding affinities (Ki values) were 2.3, 5.5, and 13.0 microM for chloroquine, quinine, and quinidine, respectively. Block by quinine was found to be only slightly voltage-dependent, thus precluding open-channel block as the main mechanism of interaction of quinine with alpha9alpha10 nAChRs. The present results add to the pharmacological characterization of alpha9alpha10-containing nicotinic receptors and indicate that the efferent olivocochlear system that innervates the cochlear hair cells is a target of these ototoxic antimalarial compounds.     

Nicotinic acetylcholine receptors in the rat stomach: I. (-)-[3H]nicotine binding

The nicotinic acetylcholine receptors in the rat stomach were characterized by means of a radioligand binding assay with (-)-[3H]nicotine as ligand. Saturation binding studies on the gastric fundus membranes revealed the presence of two binding sites with dissociation constant (KD) values of 3.1 and 289 nM, and maximum binding capacity (Bmax) values of 3.6 and 76 fmol/mg protein, respectively. The Bmax of the high affinity binding site was greatest in the cardia, followed by fundal mucosa, fundal muscle, and, finally antrum. The IC50 values of cholinergic drugs to inhibit (-)-[3H]nicotine binding in fundus membranes were as follows: (-)nicotine, 0.12 nM; cytosine, 9.3 nM; acetylcholine, 17.7 nM; carbachol, 700 nM; hexamethonium, 2270 nM. The IC50 values of alpha-bungarotoxin, d-tubocurarine and atropine were greater than 100 microM. The muscarinic acetylcholine receptors were also characterized with [3H]quinuclidinyl benzilate and the choline acetyltransferase activity was measured. These results suggest that nicotinic acetylcholine receptors as well as muscarinic acetylcholine receptors are present in the rat stomach and that the regional distribution of these receptors is uneven.     

The cholinergic pharmacology of tetrahydroaminoacridine in vivo and in vitro.

1. The effect of tetrahydroaminoacridine (THA) on cholinergically mediated behaviour in the rat and mouse has been investigated. In addition the actions of this compound on cholinesterase activity and on muscarinic and nicotinic receptors has also been examined. 2. Administration of THA (5-20 mg kg-1, i.p.) produced a dose-dependent increase in tremor, hypothermia and salivation in both rats and mice. A similar profile of activity was seen following physostigmine (0.1-0.6 mg kg-1) administration. 3. THA was approximately fifty fold less potent than physostigmine in inducing behavioural change but its effects persisted for over twice as long as those of physostigmine. For example THA-induced hypothermia was still present at 4 h in the mouse and 8 h in the rat. 4. In vitro THA was a potent non-competitive inhibitor of rat brain cholinesterase (IC50: 57 +/- 6 nM) and bovine erythrocyte acetylcholinesterase (IC50: 50 +/- 10 nM) but was a more potent inhibitor of horse serum butyrylcholinesterase (IC50: 7.2 +/- 1.4 nM). 5. Radioligand binding studies indicated that THA binds non-selectively but with moderate potency to both M1 (Ki: 600 nM) and M2 (Ki: 880 nM) muscarinic receptors. THA also interacted with the allosteric site present on cardiac M2 receptors. 6. It is concluded that THA is a reversible non-competitive inhibitor of cholinesterase with a long half life (compared with physostigmine). It also may antagonize muscarinic receptors at high doses. The long half life may account for its reported efficacy in the treatment of Alzheimer's disease.     

Interactions of atropine with heterologously expressed and native alpha 3 subunit-containing nicotinic acetylcholine receptors

(1) Atropine, a classical muscarinic antagonist, has been reported previously to inhibit neuronal nicotinic acetylcholine receptors (nAChRs). In the present study, the action of atropine has been examined on alpha3beta4 receptors expressed heterologously in Xenopus oocytes and native nAChRs in medial habenula neurons. (2) At concentrations of atropine often used to inhibit muscarinic receptors (1 micro M), responses induced by near-maximal nicotine concentrations (100 micro M) at negative holding potentials (-65 mV) are inhibited (14-30%) in a reversible manner in both alpha4 and alpha3 subunit-containing heteromeric nAChRs. Half-maximal effective concentrations (IC(50) values) for atropine inhibition are similar for the four classes of heteromeric receptors studied (4-13 micro M). (3) For alpha3beta4 nAChRs in oocytes, inhibition by atropine (10 micro M) is not overcome at higher concentrations of agonist, and is increased with membrane hyperpolarization. These results are consistent with non-competitive antagonism--possibly ion channel block. (4) At low concentrations of both nicotine (10 micro M) and atropine (<10 micro M), potentiation ( approximately 25%) of alpha3beta4 nAChR responses in oocytes is observed. The relative balance between potentiation and inhibition is dependent upon membrane potential. (5) In rat medial habenula (MHb) neurons, atropine (0.3-3.0 micro M) inhibited nicotine-induced responses in both a concentration and membrane potential-dependent manner (at -40 mV, IC(50)=4 micro M), similar to the effects on alpha3beta4-nAChRs in oocytes. However, unlike heterologously expressed receptors, potentiation was barely detectable at depolarized membrane potentials using low concentrations of nicotine (3-10 micro M). Conversely, the weak agonist, choline (1-3 mM) was observed to augment responses of MHb nAChRs.     

Muscarinic acetylcholine receptors potentiate the GABAergic transmission in the developing rat inferior colliculus

Postsynaptic currents (PSCs) were recorded using the patch-clamp technique in neurons of the rat inferior colliculus (IC) to investigate the muscarinic modulation of the GABAergic transmission. In the presence of strychnine (0.5 microM) and kynurenic acid (1 mM), spontaneous GABAergic PSCs were observed in all IC neurons investigated. Muscarine (10 microM) greatly potentiated the frequency of these GABAergic PSCs (618% of the control). 4-DAMP (50 nM), a M3 receptor preferring antagonist, greatly blocked the muscarine-evoked PSC frequency increase. The muscarinic antagonists telenzepine (50 nM; M1 preferring), methoctramine (10 microM; M2 preferring), and himbazine (10 microM; M4 preferring), and the nicotinic antagonist mecamylamine (10 microM) did not significantly affect the muscarine effect. These findings indicate that the muscarinic modulation of the GABAergic transmission is primarily mediated by M3 receptors, while M1-, M2- and M4- and nicotinic receptors do not participate substantially. In the presence of tetrodotoxin (0.5 microM), muscarine failed to increase the PSC frequency indicating that its effect depended on the generation of spikes. We suggest that GABAergic interneurones express M3 receptors at some distance from the terminal. Their activation excites GABAergic interneurones, thereby enhancing GABA release in the IC. The muscarinic modulation of the GABAergic transmission may play an important role in the maturation of inhibitory synapses in the developing IC.     

Blockade of nicotinic receptors of bovine adrenal chromaffin cells by nanomolar concentrations of atropine

Nanomolar concentrations of atropine have been considered up to now to be selective for blockade of muscarinic receptors for acetylcholine. A collateral finding indicated to us that these low concentrations of atropine could also target the neuronal nicotinic receptors. We report here a detailed study on this novel property of atropine. Catecholamine release, measured on-line with amperometry in chromaffin cells stimulated with acetylcholine pulses was blocked by atropine in a competitive manner. To corroborate a direct action of atropine on nicotinic receptors, we have employed N,N-dimethyl-N'-phenyl-piperazinium (DMPP), a pure nicotinic receptor agonist; atropine blocked its secretory responses with an IC50 of 2.04 nM. Nicotinic currents, recorded with the whole cell configuration of the patch-clamp technique were blocked by atropine in a concentration-dependent manner (IC50 of 11 nM), also showing a competitive nature. Nicotinic receptor currents in oocytes expressing bovine alpha7 and alpha3beta4 nicotinic receptors were blocked by atropine with an IC50 of 11.2 and 46.8 nM, respectively. Atropine (30 nM) also decreased the increment of the cytosolic calcium concentrations after stimulation with 30 microM DMPP in bovine chromaffin cells. However, action potentials evoked by DMPP were not modified by atropine. Our results demonstrate that nicotinic currents and their downstream consequences (i.e. cytosolic calcium elevations and catecholamine release) were blocked by nanomolar concentrations of atropine; although the blockade was partial, it must be considered when using atropine to study cholinergic neurotransmission, particularly at synapses where both nicotinic and muscarinic receptors are present i.e., the adrenal medulla and autonomic ganglia.     

Properties of muscarinic receptors mediating second messenger responses in the rabbit aorta

The second messenger response of cultured smooth muscle and endothelial cells of the rabbit aorta to acetylcholine (ACh) was investigated. ACh induced a concentration-dependent accumulation of inositol-monophosphate (InsP1) in the smooth muscle cells and a concentration-dependent reduction in basal production of adenosine 3',5'-cyclic monophosphate (cAMP) in the endothelial cells. Atropine, scopolamine and nitric oxide (NO) inhibited the ACh-induced accumulation of InsP1. The IC50 values were 55 +/- 4.3 nM, 81.2 +/- 6.5 microM and 13.3 +/- 3.5 microM, respectively. On the other hand, the inhibition of reduction in basal production of cAMP was inhibited by scopolamine (IC50 = 55.3 +/- 4.3 nM) and atropine (IC50 = 63.2 +/- 5.2 microM). Pirenzepine inhibited both the ACh-induced accumulation of InsP1 (IC50 = 1.5 +/- 0.01 microM) and reduction of basal production of cAMP (IC50 = 812 +/- 33.5 nM). However, unlike scopolamine or atropine, the M1-selective ligand was not selective to either the endothelial or smooth muscle receptors. These results demonstrate for the first time the second messenger response of the action of ACh on the endothelial muscarinic receptors and the inhibition of InsP1 formation by NO. In addition, the results also support our earlier findings that the muscarinic receptors in the endothelium and smooth muscle of the rabbit aorta can be differentiated by atropine and scopolamine, namely, the endothelial receptors have high affinity for scopolamine but extremely low affinity for atropine whilst the reverse holds true for the smooth muscle receptors.     

Antinociceptive and toxic effects of (+)-epibatidine oxalate attributable to nicotinic agonist activity.

1. Epibatidine is an analgesic substance, isolated from the skin of the poisonous frog Epipedobates tricolor, for which the mechanism of action was previously unknown. 2. The IC50 of synthetic (+)-epibatidine oxalate (the naturally occurring isomer) for [3H]-nicotine binding to rat whole-brain membranes was 0.1 nM. The (-)-isomer also exhibited high affinity (IC50 = 0.2 nM). 3. (+)- and (-)-Epibatidine exhibited much lower affinity for displacement of the muscarinic ligand [3H]-N-methylscopolamine binding to rat cortical membranes (Kapp = 6.9 microM and 16.0 microM respectively). The (+)-enantiomer of epibatidine had an antagonist/agonist (NMS/oxo-M) binding ratio of 4.2 This is consistent with a muscarinic antagonist profile. 4. (+)-Epibatidine oxalate (10 microM) did not cause significant (> 30%) displacement of radioligand binding to opioid, excitatory amino acid, benzodiazepine, 5-HT, dopamine, adrenaline or peptide receptors. 5. (+)- and (-)-Epibatidine (5-20 micrograms kg-1 s.c.) doubled response latency in the mouse hot-plate test. Antinociception and behavioural depression induced by (+)-epibatidine (5 micrograms kg-1) was fully blocked by the nicotinic antagonists mecamylamine (2 mg kg-1 s.c.) or dihydro-beta-erythroidine (2 mg kg-1 s.c.). The muscarinic antagonist scopolamine (0.4 and 10 mg kg-1 s.c.) caused partial reversal of antinociception induced by (+)-epibatidine in mice, but not in rats. 6. These findings demonstrate that (+)-epibatidine oxalate salt is a highly selective and potent nicotinic analgesic agent.     

Interaction of the hexamethonium derivative W84, an allosteric effector at muscarinic cholinoreceptors, with rat brain nicotinic binding sites

The hexamethonium derivative W84 (hexamethylene-bis-[dimethyl-(3-phthalimidopropyl)-ammonium bromide]) combined with atropine has an overadditive protective action against organophosphorus intoxications. It affects allosterically the binding of (-) [3H]N-methylscopolamine [(3H]NMS) to muscarinic cholinoceptors. Because nicotinic receptors are involved in organophosphorus intoxications, the interaction of W84 with nicotinic cholinoceptors was investigated. (-) [3H]nicotine (2.5 nM) was used to label nicotinic binding sites in rat brain membranes in 50 nM Tris, pH 7.3 at 23 degrees. Under control conditions, (-) [3H]nicotine-binding revealed a KD of 4 X 10(-9) M and a Bmax of 53 fmol/mg membrane protein. W84 inhibited (- ) [3H]nicotine-binding with an IC50 of 3 X 10(-5) M by reducing the binding affinity. The IC50 of hexamethonium was 20 X 10(-5) M. At 10(-4) M, W84 did not affect the dissociation rate of (-)[3H]nicotine, suggesting a lack of allosteric activity. For sake of comparison, the action of W84 was checked on [3H]NMS-binding (control: KD approximately 1 X 10(-9) M, Bmax approximately 500 fmol/mg prot). W84 inhibited the binding of [3H]NMS (0.5 nM) with an IC50 of 1.5 X 10(-9) M. At 10(-4) M, W84 prevented [3H]NMS-dissociation almost completely, thus displaying the allosteric action at muscarinic cholinoceptors. In conclusion, the results of the (-)[3H]nicotine-binding experiments point to a pure competitive action of W84 at nicotine cholinoceptors, lacking any allosteric effect. This competitive action may contribute to the protective effect of W84 in organophosphorus poisoning.     

In vitro effects of chlorpyrifos, parathion, methyl parathion and their oxons on cardiac muscarinic receptor binding in neonatal and adult rats.

Organophosphorus insecticides elicit toxicity by inhibiting acetylcholinesterase. Young animals are generally more sensitive than adults to these toxicants. A number of studies reported that some organophosphorus agents also bind directly to muscarinic receptors, in particular the m(2) subtype, in tissues from adult rats. As both the density and agonist affinity states of cardiac muscarinic receptors (primarily m(2)) have been reported to change in an age-related manner, we evaluated the relative in vitro sensitivity of cardiac muscarinic receptors in tissues from neonatal (7-11 days of age) and adult (90 days of age) rats to selected organophosphorus compounds (chlorpyrifos, parathion, methyl parathion and their oxygen analogs or oxons). The effects of the cholinergic agonist carbachol (100 pM-5 microM) or an organophosphorus toxicant (50 pM-10 microM) on muscarinic receptor binding were determined using the nonselective muscarinic ligand [3H]quinuclidinyl benzilate or the m(2)-preferential ligand [3H]oxotremorine-M acetate. Carbachol displaced [3H]oxotremorine labeling in adult and neonatal membranes in a relatively similar manner (IC(50)=7-20 nM). The oxons all displaced [3H]oxotremorine binding in a concentration-dependent manner, with chlorpyrifos oxon being the most potent (IC(50): neonates, 15 nM; adults, 7 nM) and efficacious (maximum displacement: neonates, 42%; adults, 56%). Interestingly, methyl parathion was an extremely potent displacer of [3H]oxotremorine binding in adult tissues (IC(50)=0.5 nM, maximum displacement=37%) but had no effect in neonatal tissues. The displacement of [3H]oxotremorine binding by chlorpyrifos oxon (10 microM) was still apparent after washing the tissues, suggesting the oxon irreversibly blocked agonist binding to the receptor while interaction with MePS appeared reversible. As effective concentrations of the oxons were relatively similar to their anticholinesterase potencies, these findings suggest that direct interaction with cardiac muscarinic receptors by some organophosphorus agents may occur at relevant exposure levels and contribute to cardiac toxicity.     

INTERACTION OF CHOLINE WITH NICOTINIC AND MUSCARINIC CHOLINERGIC RECEPTORS IN THE RAT BRAIN IN VITRO

The ability of choline to interact with nicotinic receptors was investigated by measuring its ability to inhibit the specific binding of [3H]-nicotine in rat brain. Choline, with an IC50 of 241 mumol/l, was three times more potent than its analogue deanol and almost 1000-fold less potent than acetylcholine. Choline also inhibited the binding of the antagonist [3H]-quinuclidinyl benzilate (IC50 = 2.5 mmol/l) and of the agonist [3H]-oxotremorine-M (IC50 = 165 mumol/l) to muscarinic cholinergic receptors. These results indicate that choline is able to interact directly, in vitro with brain cholinergic receptors of both the nicotinic and muscarinic type.     

Analogs of alpha-conotoxin MII are selective for alpha6-containing nicotinic acetylcholine receptors

Neuronal nicotinic acetylcholine receptors (nAChRs) both mediate direct cholinergic synaptic transmission and modulate synaptic transmission by other neurotransmitters. Novel ligands are needed as probes to discriminate among structurally related nAChR subtypes. Alpha-conotoxin MII, a selective ligand that discriminates among a variety of nAChR subtypes, fails to discriminate well between some subtypes containing the closely related alpha3 and alpha6 subunits. Structure-function analysis of alpha-conotoxin MII was performed in an attempt to generate analogs with preference for alpha6-containing [alpha6(*) (asterisks indicate the possible presence of additional subunits)] nAChRs. Alanine substitution resulted in several analogs with decreased activity at alpha3(*) versus alpha6(*) nAChRs heterologously expressed in Xenopus laevis oocytes. From the initial analogs, a series of mutations with two alanine substitutions was synthesized. Substitution at His9 and Leu15 (MII[H9A;L15A]) resulted in a 29-fold lower IC(50) at alpha6beta4 versus alpha3beta4 nAChRs. The peptide had a 590-fold lower IC(50) for alpha6/alpha3beta2 versus alpha3beta2 and a 2020-fold lower IC(50) for alpha6/alpha3beta2beta3 versus alpha3beta2 nAChRs. MII[H9A;L15A] had little or no activity at alpha2beta2, alpha2beta4, alpha3beta4, alpha4beta2, alpha4beta4, and alpha7 nAChRs. Functional block by MII[H9A;L15A] of rat alpha6/alpha3beta2beta3 nAChRs (IC(50) = 2.4 nM) correlated well with the inhibition constant of MII[H9A;L15A] for [(125)I]alpha-conotoxin MII binding to putative alpha6beta2(*) nAChRs in mouse brain homogenates (K(i) = 3.3 nM). Thus, structure-function analysis of alpha-conotoxin MII enabled the creation of novel selective antagonists for discriminating among nAChRs containing alpha3 and alpha6 subunits.     

Tolterodine and its active 5-hydroxymethyl metabolite: pure muscarinic receptor antagonists

Tolterodine and its major active 5-hydroxymethyl metabolite (5-HM) are potent muscarinic receptor antagonists that show selectivity for the urinary bladder over salivary glands in vivo. This tissue selectivity cannot be attributed to muscarinic receptor subtype selectivity, since both compounds are non-selective with respect to the M1-M5 receptor subtypes. The aim of the present in vitro study was to determine the specificity of tolterodine and 5-HM for muscarinic receptors compared to other potential cellular targets. Carbachol-induced contractions of isolated guinea pig bladder were effectively inhibited by tolterodine (IC50 14 nM) and 5-HM (IC50 5.7 nM). Tolterodine and 5-HM were weak inhibitors of effects mediated by alpha-adrenergic receptors (rat portal vein), histamine receptors (guinea pig ileum) and calcium channels (guinea pig potassium-depolarised urinary bladder, spontaneously beating right atria and electrically-driven right papillary muscle). The IC50 values were in the microM range and the antimuscarinic potency of tolterodine was 27, 200 and 370-485 times higher, respectively, than its potency in blocking histamine receptors, alpha-adrenoceptors and calcium channels. The active metabolite, 5-HM, was >900 times less potent at these sites than at bladder muscarinic receptors. Radioligand binding data on 54 different receptors and binding sites showed that tolterodine and 5-HM bind with significant affinity only at muscarinic receptors. In conclusion, the results of the present study indicate that both tolterodine and 5-HM are specific muscarinic receptor antagonists. The tissue selectivity of these agents in vivo cannot therefore be explained by secondary pharmacological actions.     

Nicotinic acetylcholine receptors and imaging

In vivo imaging techniques like positron emission tomography (PET) and single photon emission computed tomography (SPECT) offer the possibility to monitor human central nicotinic acetylcholine receptors (nAChRs) in a variety of central nervous system disorders. In the past, the only available PET radiotracer for imaging nAChRs in the human brain, [11C]-(-)-nicotine, suffered from a spectrum of not suitable properties for in vivo imaging. Current efforts are focused on the development of new, highly specific and highly selective radioligands based on different structural classes (e.g. nicotine, epibatidine, 3-pyridyl ether analogues) for central nAChRs. The most promising compounds are halogenated 3-pyridyl ether compounds for imaging alpha 4 beta 2 nAChRs. But there is still a lack for radiotracers for other subtypes of nicotinic acetylcholine receptors being a promising area of interest.     

Ketamine and its preservative, benzethonium chloride, both inhibit human recombinant alpha7 and alpha4beta2 neuronal nicotinic acetylcholine receptors in Xenopus oocytes

1. Ketamine is a dissociative anaesthetic that is formulated as Ketalar, which contains the preservative benzethonium chloride (BCl). We have studied the effects of pure racemic ketamine, the preservative BCl and the Ketalar mixture on human neuronal nicotinic acetylcholine receptors (nAChRs) composed of the alpha7 subunit or alpha4 and beta2 subunits expressed in Xenopus laevis oocytes. 2. Ketamine inhibited responses to 1 mM acetylcholine (ACh) in both the human alpha7 and alpha4beta2 nAChRs, with IC(50) values of 20 and 50 microM respectively. Inhibition of the alpha7 nAChRs occurred within a clinically relevant concentration range, while inhibition of the alpha4beta2 nAChR was observed only at higher concentrations. The Ketalar formulation inhibited nAChR function more effectively than was expected given its ketamine concentration. The surprising increased inhibitory potency of Ketalar compared with pure ketamine appeared to be due to the activity of BCl, which inhibited both alpha7 (IC(50) value of 122 nM) and alpha4beta2 (IC(50) value of 49 nM) nAChRs at concentrations present in the clinical formulation of Ketalar. 3. Ketamine is a noncompetitive inhibitor at both the alpha7 and alpha4beta2 nAChR. In contrast, BCl causes a parallel shift in the ACh dose-response curve at the alpha7 nAChR suggesting competitive inhibition. Ketamine causes both voltage-dependent and use-dependent inhibition, only in the alpha4beta2 nAChR. 4. Since alpha7 nAChRs are likely to be inhibited during clinical use of Ketalar, the actions of ketamine and BCl on this receptor subtype may play a role in the profound analgesia, amnesia, immobility and/or autonomic modulation produced by this anaesthetic.     

N-n-alkylnicotinium analogs, a novel class of antagonists at α4β2* Nicotinic acetylcholine receptors: Inhibition of S(-)-nicotine-evoked 86Rb+Efflux from rat thalamic synaptosomes

Pyridine N-n-alkylation of S(-)-nicotine (NIC) affords N-n-alkylnicotinium analogs, previously shown to competitively inhibit [(3)H]NIC binding and interact with alpha4beta2* nicotinic receptors (nAChRs). The present study determined the ability of the analogs to inhibit NIC-evoked (86)Rb(+) efflux from rat thalamic synaptosomes to assess functional interaction with alpha4beta2* nAChRs. In a concentration-dependent manner, NIC evoked (86)Rb(+) efflux (EC(50) = 170 nmol/L). Analog-induced inhibition of NIC-evoked (86)Rb(+) efflux varied over a approximately 450-fold range. Analogs with long n-alkyl chain lengths (C(9)-C(12)) inhibited efflux in the low nmol/L range (IC(50) = 9-20 nmol/L), similar to dihydro-beta-erythroidine (IC(50) = 19 nmol/L). Compounds with shorter n-alkyl chain lengths (C(1)-C(8)) produced inhibition in the low micromol/L range (IC(50) = 3-12 micromol/L). C(10) and C(12) analogs completely inhibited NIC-evoked efflux, whereas C(1-9) analogs produced maximal inhibition of only 10% to 60%. While the C(10) analog N-n-decylnicotinium iodide (NDNI) did not produce significant inhibition of NIC-evoked dopamine release in previously reported studies, NDNI possesses high affinity for [(3)H]NIC binding sites (K(i) = 90 nmol/L) and is a potent and efficacious inhibitor of NIC-evoked (86)Rb(+) efflux as demonstrated in the current studies. Thus, NDNI is a competitive, selective antagonist at alpha4beta2* nAChRs.     

Direct interaction of serotonin type 3 receptor ligands with recombinant and native alpha 9 alpha 10-containing nicotinic cholinergic receptors

In the present work, we characterized the effects of serotonin type 3 receptor ligands on recombinant and native alpha 9 alpha 10-containing nicotinic acetylcholine receptors (nAChRs). Our results indicate that the recombinant alpha 9 alpha 10 nAChR shares striking pharmacological properties with 5-HT(3) ligand-gated ion channels. Thus, 5-HT(3) receptor antagonists block ACh-evoked currents in alpha 9 alpha 10-injected Xenopus laevis oocytes with a rank order of potency of tropisetron (IC(50), 70.1 +/- 0.9 nM) > ondansetron (IC(50), 0.6 +/- 0.1 microM) = MDL 72222 (IC(50), 0.7 +/- 0.1 microM). Although serotonin does not elicit responses in alpha 9 alpha 10-injected oocytes, it blocks recombinant alpha 9 alpha 10 receptors in a noncompetitive and voltage-dependent manner (IC(50), 5.4 +/- 0.6 microM). On the other hand, we demonstrate an in vivo correlate of these properties of the recombinant receptor, with those of the alpha 9 alpha 10-containing nAChR of frog saccular hair cells. The possibility that the biogenic amine serotonin might act as a neuromodulator of the cholinergic efferent transmission in the vestibular apparatus and in the organ of Corti is discussed.     

Chemical and functional identification and characterization of novel sulfated alpha-conotoxins from the cone snail Conus anemone

An LC/MS analysis with diagnostic screening for the detection of peptides with posttranslational modifications revealed the presence of novel sulfated peptides within the alpha-conotoxin molecular mass range in Conus anemone crude venom. A functional assay of the extract showed activity at several neuronal nicotinic acetylcholine receptors (nAChRs). Three sulfated alpha-conotoxins (AnIA, AnIB, and AnIC) were identified by LC/MS and assay-directed fractionation and sequenced after purification. The most active of these, alpha-AnIB, was further characterized and used to investigate the influence of posttranslational modifications on affinity. Synthetic AnIB exhibited subnanomolar potency at the rat alpha3beta2 nAChR (IC50 0.3 nM) and was 200-fold less active on the rat alpha7 nAChR (IC50 76 nM). The unsulfated peptide [Tyr16]AnIB showed a 2-fold and 10-fold decrease in activities at alpha3beta2 (IC50 0.6 nM) and alpha7 (IC50 836 nM) nAChR, respectively. Likewise, removal of the C-terminal amide had a greater influence on potency at the alpha7 (IC50 367 nM) than at the alpha3beta2 nAChR (IC50 0.5 nM). Stepwise removal of two N-terminal glycine residues revealed that these residues affect the binding kinetics of the peptide. Comparison with similar 4/7-alpha-conotoxin sequences suggests that residue 11 (alanine or glycine) and residue 14 (glutamine) constitute important determinants for alpha3beta2 selectivity, whereas the C-terminal amidation and sulfation at tyrosine-16 favor alpha7 affinity.     

Neuropharmacology of the muscarinic antagonist telenzepine in myenteric ganglia of the guinea-pig small intestine

Intracellular recording methods were used to investigate the actions of the putative M1 muscarinic receptor antagonist telenzepine on the electrical and synaptic behavior of myenteric neurons. Telenzepine had no effect on resting membrane potential, input resistance, excitability and antidromic potentials in both AH/type 2 and S/type 1 neurons, when applied in concentrations of 0.1-2000 nM, although higher concentrations (10-100 microM) did have a significant non-specific effect on the postsynaptic membrane. Micromolar concentrations of telenpzepine (1-2 microM) had no effect on excitatory responses to substance P, vasoactive intestinal peptide, the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium or the nicotinic action of acetylcholine. Nicotinic fast excitatory postsynaptic potentials were also unaffected by 2 microM telenzepine. In contrast, at submicromolar concentrations (100 nM), telenzepine abolished responses to either muscarine or the muscarinic component of the acetylcholine response. The excitatory effect of muscarine at postsynaptic M1 receptors was dose dependently inhibited by telenzepine (0.1-1000 nM) at concentrations which had no effect on the electrical properties of the cells. This effect was slowly reversible, usually requiring more than 60 min for significant recovery. The threshold dose of telenzepine as an antagonist of the muscarinic depolarization in AH/type 2 neurons was in the range of 0.1-1 nM. The IC50 concentration of telenzepine needed to abolish the response was 8.5 nM. A small proportion of stimulus-evoked slow excitatory postsynaptic potentials in both AH/type 2 and S/type 1 cells were abolished by 1 microM telenzepine, while the majority of them remained unaffected, indicating that some slow excitatory postsynaptic potentials are mediated by the muscarinic action of released acetylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)