Bupropion inhibits nicotine-evoked [(3)H]overflow from rat striatal slices preloaded with [(3)H]dopamine and from rat hippocampal slices preloaded with [(3)H]norepinephrine

Bupropion, an efficacious antidepressant and smoking cessation agent, inhibits dopamine and norepinephrine transporters (DAT and NET, respectively). Recently, bupropion has been reported to noncompetitively inhibit alpha3beta2, alpha3beta4, and alpha4beta2 nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes or established cell lines. The present study evaluated bupropion-induced inhibition of native alpha3beta2* and alpha3beta4* nAChRs using functional neurotransmitter release assays, nicotine-evoked [(3)H]overflow from superfused rat striatal slices preloaded with [(3)H]dopamine ([(3)H]DA), and nicotine-evoked [(3)H]overflow from hippocampal slices preloaded with [(3)H]norepinephrine ([(3)H]NE). The mechanism of inhibition was evaluated using Schild analysis. To eliminate the interaction of bupropion with DAT or NET, nomifensine or desipramine, respectively, was included in the superfusion buffer. A high bupropion concentration (100 microM) elicited intrinsic activity in the [(3)H]DA release assay. However, none of the concentrations (1 nM-100 microM) examined evoked [(3)H]NE overflow and, thus, were without intrinsic activity in this assay. Moreover, bupropion inhibited both nicotine-evoked [(3)H]DA overflow (IC(50) = 1.27 microM) and nicotine-evoked [(3)H]NE overflow (IC(50) = 323 nM) at bupropion concentrations well below those eliciting intrinsic activity. Results from Schild analyses suggest that bupropion competitively inhibits nicotine-evoked [(3)H]DA overflow, whereas evidence for receptor reserve was obtained upon assessment of bupropion inhibition of nicotine-evoked [(3)H]NE overflow. Thus, bupropion acts as an antagonist at alpha3beta2* and alpha3beta4* nAChRs in rat striatum and hippocampus, respectively, across the same concentration range that inhibits DAT and NET function. The combination of nAChR and transporter inhibition produced by bupropion may contribute to its clinical efficacy as a smoking cessation agent.     

N-n-alkylpyridinium analogs,a novel class of nicotinic receptor antagonists: selective inhibition of nicotine-evoked [3H] dopamine overflow from superfused rat striatal slices

Structural simplification of N-n-alkylnicotinium analogs, antagonists at neuronal nicotinic acetylcholine receptors (nAChRs), was achieved by removal of the N-methylpyrrolidino moiety affording N-n-alkylpyridinium analogs with carbon chain lengths of C1 to C20. N-n-Alkylpyridinium analog inhibition of [3H]nicotine and [3H]methyllycaconitine binding to rat brain membranes assessed interaction with alpha4beta2* and alpha7* nAChRs, respectively, whereas inhibition of nicotine-evoked 3H overflow from [3H]dopamine ([3H]DA)-preloaded rat striatal slices assessed antagonist action at nAChR subtypes mediating nicotine-evoked DA release. No inhibition of [3H]methyllycaconitine binding was observed, although N-n-alkylpyridinium analogs had low affinity for [3H]nicotine binding sites, i.e., 1 to 3 orders of magnitude lower than that of the respective N-n-alkylnicotinium analogs. These results indicate that the N-methylpyrrolidino moiety in the N-n-alkylnicotinium analogs is a structural requirement for potent inhibition of alpha4beta2* nAChRs. Importantly, N-n-alkylpyridinium analogs with n-alkyl chains < C10 did not inhibit nicotine-evoked [3H]DA overflow, whereas analogs with n-alkyl chains ranging from C10 to C20 potently and completely inhibited nicotine-evoked [3H]DA overflow (IC50 = 0.12-0.49 microM), with the exceptions of N-n-pentadecylpyridinium bromide (C15) and N-n-eicosylpyridinium bromide (C20), which exhibited maximal inhibition of approximately 50%. The mechanism of inhibition of a representative analog of this structural series, N-n-dodecylpyridinium iodide, was determined by Schild analysis. Linear Schild regression with slope not different from unity indicated competitive antagonism at nAChRs mediating nicotine-evoked [3H]DA overflow and a KB value of 0.17 microM. Thus, the simplified N-n-alkylpyridinium analogs are potent, selective, and competitive antagonists of nAChRs mediating nicotine-evoked [3H]DA overflow, indicating that the N-methylpyrrolidino moiety is not a structural requirement for interaction with nAChR subtypes mediating nicotine-evoked DA release.     

Effect of novel alpha-conotoxins on nicotine-stimulated [3H]dopamine release from rat striatal synaptosomes

Nicotine's action on the midbrain dopaminergic neurons is mediated by nicotinic acetylcholine receptors (nAChRs) that are present on the cell bodies and the terminals of these neurons. Previously, it was suggested that one of the nAChR subtypes located on striatal dopaminergic terminals may be an alpha3beta2 subtype, based on partial inhibition of nicotine-stimulated [(3)H]dopamine release by alpha-conotoxin MII, a potent inhibitor of heterologously expressed alpha3beta2 nAChRs. More recent studies indicated that alpha-conotoxin MII also potently blocks alpha6-containing nAChRs. In the present study, we have examined the nAChR subtype(s) modulating [(3)H]dopamine release from striatal terminals by using novel alpha-conotoxins that have 37- to 78-fold higher selectivity for alpha6-versus alpha3-containing nAChRs. All of the peptides partially (20-35%) inhibit nicotine-stimulated [(3)H]dopamine release with IC(50) values consistent with those obtained with heterologously expressed rat alpha6-containing nicotinic acetylcholine receptors. These results, together with previous studies by others, further support the idea that alpha6-containing nicotinic receptors modulate nicotine-stimulated dopamine release from rat striatal synaptosomes.     

N,N'-Alkane-diyl-bis-3-picoliniums as nicotinic receptor antagonists: inhibition of nicotine-evoked dopamine release and hyperactivity

The current study evaluated a new series of N,N'-alkane-diyl-bis-3-picolinium (bAPi) analogs with C6-C12 methylene linkers as nicotinic acetylcholine receptor (nAChR) antagonists, for nicotine-evoked [3H]dopamine (DA) overflow, for blood-brain barrier choline transporter affinity, and for attenuation of discriminative stimulus and locomotor stimulant effects of nicotine. bAPi analogs exhibited little affinity for alpha4beta2* (* indicates putative nAChR subtype assignment) and alpha7* high-affinity ligand binding sites and exhibited no inhibition of DA transporter function. With the exception of C6, all analogs inhibited nicotine-evoked [3H]DA overflow (IC50 = 2 nM-6 microM; Imax = 54-64%), with N,N'-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB; C12) being most potent. bPiDDB did not inhibit electrically evoked [3H]DA overflow, suggesting specific nAChR inhibitory effects and a lack of toxicity to DA neurons. Schild analysis suggested that bPiDDB interacts in an orthosteric manner at nAChRs mediating nicotine-evoked [3H]DA overflow. To determine whether bPiDDB interacts with alpha-conotoxin MII-sensitive alpha6beta2-containing nAChRs, slices were exposed concomitantly to maximally effective concentrations of bPiDDB (10 nM) and alpha-conotoxin MII (1 nM). Inhibition of nicotine-evoked [3H]DA overflow was not different with the combination compared with either antagonist alone, suggesting that bPiDDB interacts with alpha6beta2-containing nAChRs. C7, C8, C10, and C12 analogs exhibited high affinity for the blood-brain barrier choline transporter in vivo, suggesting brain bioavailability. Although none of the analogs altered the discriminative stimulus effect of nicotine, C8, C9, C10, and C12 analogs decreased nicotine-induced hyperactivity in nicotine-sensitized rats, without reducing spontaneous activity. Further development of nAChR antagonists that inhibit nicotine-evoked DA release and penetrate brain to antagonize DA-mediated locomotor stimulant effects of nicotine as novel treatments for nicotine addiction is warranted.     

Antidepressant drugs with varying pharmacological profiles alter rat pineal beta adrenergic-mediated function

The effects of acute and chronic administrations of two antidepressant drugs with differing pharmacological profiles on pineal beta adrenergic receptor-mediated functions were examined in the rat. Animals were treated with control powdered food or with either imipramine or iprindole-containing diets (0.067%, w/w) for various time intervals. Animals were sacrificed during different phases of the light/dark cycle and pineal [3H]dihydroalprenolol (DHA) binding, N-acetyltransferase (NAT) activity, N-acetylserotonin (NAS) and melatonin levels were measured. Plasma drugs and metabolite concentrations were also assessed. A 3-day treatment with imipramine resulted in an unchanged pineal [3H] DHA binding and an increase in pineal serotonin (5-HT), NAS and melatonin. A comparable treatment with iprindole did not alter any pineal measures. Three weeks of imipramine treatment resulted in therapeutic plasma drug and metabolite concentrations and elicited a reduction (31%) in the density of pineal [3H] DHA binding. This treatment, in addition, suppressed the dark-induced activation of the intracellular enzyme NAT (38%) and the concentrations of NAS (25%) and melatonin (23%) without altering pineal 5-HT rhythm. No apparent shift was observed in the NAT, NAS and melatonin rhythms. Chronic treatment with the atypical antidepressant iprindole for 3 weeks resulted in-plasma iprindole concentrations of 76 ng/ml and a significant reduction (24%) in pineal 5-HT levels 5 hr into the dark phase of the light/dark cycle. Pineal NAT activity and NAS and melatonin content were not significantly reduced by this treatment. However, 4 weeks of iprindole ingestion produced plasma drug concentrations of 141 ng/ml and significantly reduced pineal [3H] DHA binding density (18%) without changing binding affinity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of nicotinic agonist-induced [(3)H]dopamine release from synaptosomes prepared from four mouse brain regions

The inhibition of uptake of [(3)H]dopamine into synaptosomes prepared from four mouse brain regions was investigated. The inhibition curves demonstrated that in three regions, striatum, nucleus accumbens, and olfactory tubercle, [(3)H]dopamine was taken up exclusively by dopaminergic terminals. In frontal cortex, however, only a portion of the uptake was into dopaminergic terminals, with a larger amount taken up by noradrenergic terminals, and another small portion by serotonergic terminals. Release studies in frontal cortex indicated that in this region only dopaminergic and noradrenergic terminals are capable of packaging [(3)H]dopamine in a form allowing vesicle-mediated release; additionally, only the dopaminergic terminals have functional presynaptic nAChRs that, when stimulated by nicotinic agonists, evoke [(3)H]dopamine release. Agonist-stimulated [(3)H]dopamine release was characterized from synaptosomes prepared from four mouse brain regions. alpha-Conotoxin MII was a partial inhibitor of dopamine release in all of the brain regions, which suggests that a minimum of two nicotinic cholinergic receptors (nAChRs) are expressed in the nerve terminals of all four brain regions. No nicotine-induced [(3)H]dopamine release was detected in any brain region when the synaptosomes were prepared from beta2 null mutant mice, which indicates that the beta2 subunit is required for all nAChRs mediating this release. Dose-response curves were constructed for seven agonists in each of the brain regions. The pharmacological properties of synaptosomal [(3)H]dopamine release appear similar across the four brain regions. The results suggest that all four regions express the same nAChRs, although subtle regional differences may exist.     

Reboxetine: functional inhibition of monoamine transporters and nicotinic acetylcholine receptors

The present study determined whether repeated administration of the antidepressant and selective norepinephrine (NE) uptake inhibitor reboxetine resulted in an adaptive modification of the function of the NE transporters (NETs), serotonin (5-HT) transporters, or dopamine (DA) transporters. Because antidepressants may be effective tobacco smoking cessation agents and because antidepressants have recently been shown to interact with nicotinic acetylcholine receptors (nAChRs), the interaction of reboxetine with nAChRs was also evaluated. Repeated administration of reboxetine (10 mg/kg i.p., twice daily for 14 days) did not alter the potency or selectivity of reboxetine inhibition of [(3)H]NE, [(3)H]DA, or [(3)H]5-HT uptake into striatal or hippocampal synaptosomes (IC(50) values = 8.5 nM, 89 microM, and 6.9 microM, respectively). In a separate series of experiments, reboxetine did not inhibit (K(i) > 1 microM) [(3)H]methyllycaconitine, [(3)H]cytisine, or [(3)H]epibatidine binding to rat whole brain membranes. However, at concentrations that did not exhibit intrinsic activity, reboxetine potently inhibited (IC(50) value = 7.29 nM) nicotine-evoked [(3)H]NE overflow from superfused hippocampal slices via a noncompetitive mechanism. In the latter experiments, the involvement of NET was eliminated by inclusion of desipramine (10 microM) in the superfusion buffer. Reboxetine also inhibited (IC(50) value = 650 nM) nicotine-evoked (86)Rb(+) efflux at reboxetine concentrations that did not exhibit intrinsic activity in this assay. Thus, in addition to inhibition of NET function, reboxetine inhibits nAChR function, suggesting that it may have potential as a smoking cessation agent.     

Chronic nicotine differentially regulates alpha6- and beta3-containing nicotinic cholinergic receptors in rat brain

We investigated the effects of chronic nicotine on alpha6- and beta3-containing nicotinic acetylcholine receptors (nAChRs) in two rat brain regions using three methodological approaches: radioligand binding, immunoprecipitation, and nicotine-stimulated synaptosomal release of dopamine. Nicotine was administered by osmotic minipumps for 2 weeks. Quantitative autoradiography with [(125)I]alpha-conotoxin MII to selectively label alpha6(*) nAChRs showed a 28% decrease in binding in the striatum but no change in the superior colliculus. Immunoprecipitation of nAChRs labeled by [(3)H]epibatidine in these two regions showed that chronic nicotine increased alpha4- and beta2-containing nAChRs by 39 to 67%. In contrast, chronic nicotine caused a 39% decrease in alpha6-containing nAChRs in striatum but no change in superior colliculus. No changes in beta3-containing nAChRs were seen in either region after chronic nicotine. The decreased expression of alpha6-containing nAChRs persisted for at least 3 days, recovering to baseline by 7 days after removal of the pumps. There was a small but significant decrease in total nicotine-stimulated dopamine release in striatal synaptosomes after nicotine exposure. However, the component of dopamine release that was resistant to alpha-conotoxin MII blockade was unaffected, whereas dopamine release that was sensitive to blockade by alpha-conotoxin MII was decreased by 56%. These findings indicate that the alpha6(*) nAChR is regulated differently from other nAChR subtypes, and they suggest that the inclusion of a beta3 subunit with alpha6 may serve to inhibit nicotine-induced down-regulation of these receptors.     

Cotinine selectively activates a subpopulation of alpha3/alpha6beta2 nicotinic receptors in monkey striatum

The nicotine metabolite cotinine is an abundant long-lived bio-active compound that may contribute to the overall physiological effects of tobacco use. Although its mechanism of action in the central nervous system has not been extensively investigated, cotinine is known to evoke dopamine release in the nigrostriatal pathway through an interaction at nicotinic receptors (nAChRs). Because considerable evidence now demonstrates the presence of multiple nAChRs in the striatum, the present experiments were done to determine the subtypes through which cotinine exerts its effects in monkeys, a species that expresses similar densities of striatal alpha4beta2* (nAChR containing the alpha4 and beta2 subunits, but not alpha3 or alpha6) and alpha3/alpha6beta2* (nAChR composed of the alpha3 or alpha6 subunits and beta2) nAChRs. Competition binding studies showed that cotinine interacts with both alpha4beta2* and alpha3/alpha6beta2* nAChR subtypes in the caudate, with cotinine IC(50) values for inhibition of 5-[(125) I]iodo-3-[2(S)-azetinylmethoxy]pyridine-2HCl ([(125)I]A-85380) and (125)I-alpha-conotoxinMII binding in the micromolar range. This interaction at the receptor level is of functional significance because cotinine stimulated both alpha4beta2* and alpha3/alpha6beta2* nAChR [(3)H]dopamine release from caudate synaptosomes. Our results unexpectedly showed that nicotine evokes [(3)H]dopamine release from two alpha3/alpha6beta2* nAChR populations, one of which was sensitive to cotinine and the other was not. This cotinine-insensitive subtype was only present in the medial caudate and was preferentially lost with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced nigrostriatal damage. In contrast, cotinine and nicotine elicited equivalent levels of alpha4beta2* nAChR-mediated dopamine release. These data demonstrate that cotinine functionally discriminates between two alpha3/alpha6beta2* nAChRs in monkey striatum, with the cotinine-insensitive alpha3/alpha6beta2* nAChR preferentially vulnerable to nigrostriatal damage.     

Paraquat exposure reduces nicotinic receptor-evoked dopamine release in monkey striatum

Paraquat, an herbicide widely used in the agricultural industry, has been associated with lung, liver, and kidney toxicity in humans. In addition, it is linked to an increased risk of Parkinson's disease. For this reason, we had previously investigated the effects of paraquat in mice and showed that it influenced striatal nicotinic receptor (nAChR) expression but not nAChR-mediated dopaminergic function. Because nonhuman primates are evolutionarily closer to humans and may better model the effects of pesticide exposure in man, we examined the effects of paraquat on striatal nAChR function and expression in monkeys. Monkeys were administered saline or paraquat once weekly for 6 weeks, after which nAChR levels and receptor-evoked [(3)H]dopamine ([(3)H]DA) release were measured in the striatum. The functional studies showed that paraquat exposure attenuated dopamine (DA) release evoked by alpha3/alpha6beta2(*) (nAChR that is composed of the alpha3 or alpha6 subunits, and beta2; the asterisk indicates the possible presence of additional subunits) nAChRs, a subtype present only on striatal dopaminergic terminals, with no decline in release mediated by alpha4beta2(*) (nAChR containing alpha4 and beta2 subunits, but not alpha3 or alpha6) nAChRs, present on both DA terminals and striatal neurons. Paraquat treatment decreased alpha4beta2(*) but not alpha3/alpha6beta2(*) nAChR expression. The differential effects of paraquat on nAChR expression and receptor-evoked [(3)H]DA release emphasize the importance of evaluating changes in functional measures. The finding that paraquat treatment has a negative impact on striatal nAChR-mediated dopaminergic activity in monkeys but not mice indicates the need for determining the effects of pesticides in higher species.     

A further evaluation of the effects of K+ depolarization on glutamate-evoked [3H]dopamine release from striatal slices.

Exogenous glutamate will evoke dopamine (DA) release from striatal slices in vitro. To further characterize glutamate-evoked DA release from striatal slices, experiments were designed to: 1) determine if sufficient endogenous glutamate can be released in vitro to presynaptically mediate [3H]DA release in the absence of Mg++ and 2) reevaluate how K+ depolarization affects glutamate-evoked [3H]DA release. Removal of Mg++ to potentiate N-methyl-D-aspartate (NMDA) receptor-mediated DA release increased 15 mM K(+)-evoked [3H]DA release to about 200% of control. The potentiation of this release was probably not mediated by NMDA receptors because it was not blocked by the glutamate receptor antagonists MK-801, 6,7-dinitroquinoxalinedione (DNQX) or kynurenate. Furthermore, the removal of Mg++ increased DA release substantially (200%) in the presence of 5 microM sulpiride and 10 microM nomifensine, indicating that DA reuptake and DA D2 autoreceptors are not primarily responsible for increased DA release. In the absence of Mg++, depolarization produced by 20 mM or greater [K+] inhibited DA released by exogenous glutamate, whereas a much higher [K+] was necessary to evoke endogenous glutamate release. In the presence of 1.5 mM Mg++, a reduction of the "Mg++ blockade" of NMDA receptors by 15 mM K+ depolarization during glutamate-evoked DA release was evaluated with and without the DA reuptake inhibitor nomifensine and the DA D2 antagonist sulpiride. DA released by K+ depolarization (Mg++ present) was markedly increased by 1 mM glutamate, but this effect was only partially reversed by kynurenate or high concentrations of either MK-801 (25 microM) or DNQX (100 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Active transport of high-affinity choline and nicotine analogs into the central nervous system by the blood-brain barrier choline transporter

Cigarette smoking is strongly implicated in the development of cardiovascular disorders. Recently identified nicotinium analogs may have therapeutic benefit as smoking cessation therapies but may have restricted entry into the central nervous system by the blood-brain barrier (BBB) due to their physicochemical properties. Using the in situ perfusion technique, lobeline, choline, and nicotinium analogs were evaluated for binding to the BBB choline transporter. Calculated apparent K(i) values for the choline transporter were 1.7 microM N-n-octyl choline, 2.2 microM N-n-hexyl choline, 27 microM N-n-decylnicotinium iodide, 31.9 microM N-n-octylpyridinium iodide, 49 microM N-n-octylnicotinium iodide (NONI), 393 microM lobeline, and >/=1000 microM N-methylnicotinium iodide. Nicotine and N-methylpyridinium iodide, however, do not apparently interact with the BBB choline transporter. Given NONI's apparent K(i) value determined in this study and its ability to inhibit nicotine-evoked dopamine release from superfused rat brain slices, potential brain entry of NONI via the BBB choline transporter was evaluated. [(3)H]NONI exhibited a BBB transfer coefficient value of approximately 1.6 x 10(-3) ml/s/g and a K(m) of approximately 250 microM. Unlabeled choline addition to the perfusion fluid reduced [(3)H]NONI brain uptake. We hypothesize the N-n-octyl group on the pyridinium nitrogen of NONI facilitates brain entry via the BBB choline transporter. Thus, NONI may have utility as a smoking cessation agent, given its ability to inhibit nAChRs mediating nicotine-evoked dopamine release centrally, and to be distributed to brain via the BBB choline transporter.     

Carrier-mediated transport of the quaternary ammonium neuronal nicotinic receptor antagonist n,n'-dodecylbispicolinium dibromide at the blood-brain barrier

The quaternary ammonium compound N,N'-dodecyl-bispicolinium dibromide (bPiDDB) potently and selectively inhibits nicotinic receptors (nAChRs) mediating nicotine-evoked [(3)H]dopamine release and decreases nicotine self-administration, suggesting that this polar, charged molecule penetrates the blood-brain barrier (BBB). This report focuses on 1) BBB penetration of bPiDDB; 2) the mechanism of permeation; and 3) comparison of bPiDDB to the cations choline and N-octylnicotinium iodide (NONI), both of which are polar, charged molecules that undergo facilitated BBB transport. The BBB permeation of [(3)H]choline, [(3)H]NONI, and [(14)C]bPiDDB was evaluated using in situ rat brain perfusion methods. Cerebrovascular permeability surface-area product (PS) values for [(3)H]choline, [(3)H]NONI, and [(14)C]bPiDDB were comparable (1.33 +/- 0.1, 1.64 +/- 0.15, and 1.3 +/- 0.3 ml/s/g, respectively). To ascertain whether penetration was saturable, unlabeled substrate was added to the perfusion fluid. Unlabeled choline (500 microM) reduced the PS of [(3)H]choline to 0.15 +/- 0.06 microl/s/g (p < 0.01). Likewise, unlabeled bPiDDB (500 microM) reduced the PS of [(14)C]bPiDDB to 0.046 +/- 0.005 microl/s/g (p < 0.01), whereas unlabeled NONI reduced the PS for [(3)H]NONI by approximately 50% to 0.73 +/- 0.31 microl/s/g. The PS of [(14)C]bPiDDB was reduced (p < 0.05) in the presence of 500 microM choline, indicating that the BBB choline transporter may be responsible for the transport of bPiDDB into brain. Saturable kinetic parameters for [(14)C]bPiDDB were similar to those for [(3)H]choline. The current results suggest that bPiDDB uses the BBB choline transporter for approximately 90% of its permeation into brain, and they demonstrate the carrier-mediated BBB penetration of a novel bisquaternary ammonium nAChR antagonist.     

Nicotinic acetylcholine receptor-mediated [3H]dopamine release from hippocampus

The mechanism of nicotinic acetylcholine receptor (nAChR)-induced hippocampal dopamine (DA) release was investigated using rat hippocampal slices. nAChRs involved in hippocampal DA and norepinephrine (NE) release were investigated using prototypical agonists and antagonists and several relatively novel compounds: ABT-594 [(R)-5-(2-azetidinylmethoxy)-2-chloropyridine], (+/-)-UB-165 [(2-chloro-5-pyridyl)-9-azabicyclo [4.2.1]non2-ene], and MG 624 [N,N,N-triethyl-2-[4-(2 phenylethenyl)phenoxy]-ethanaminium iodine]. (+/-)-Epibatidine, (+/-)-UB-165, anatoxin-a, ABT-594, (-)-nicotine, 1,1-dimethyl-4-phenyl-piperazinium iodide, and (-)-cytisine (in decreasing order of potency) evoked [(3)H]DA release in a mecamylamine-sensitive manner. Aside from (+/-)-UB-165, all the agonists displayed full efficacy relative to 100 microM (-)-nicotine in [(3)H]DA release. In contrast, (+/-)-UB-165 was a partial agonist, evoking 58% of 100 microM (-)-nicotine response. Mecamylamine, MG 624, hexamethonium, d-tubocurare, and dihydro-beta-erythroidine (in decreasing order of potency), but not alpha-conotoxin-MII, methyllycaconitine, alpha-conotoxin-ImI, or alpha-bungarotoxin, attenuated 100 microM (-)-nicotine-evoked [(3)H]DA release in a concentration-dependent manner. (+/-)-UB-165, ABT-594, and MG 624 exhibited different pharmacologic profiles in the [(3)H]NE release assay when compared with their effect on [(3)H]DA release. ABT-594 was 4.5-fold more potent, and (+/-)-UB-165 was a full agonist in contrast to its partial agonism in [(3)H]DA release. MG 624 potently and completely blocked NE release evoked by 100 microM (-)-nicotine and 10 microM (+/-)-UB-165, whereas it only partially inhibited (-)-nicotine-evoked [(3)H]DA release. In conclusion, we provide evidence that [(3)H]DA can be evoked from the hippocampus and that the pharmacologic profile for nAChR-evoked hippocampal [(3)H]DA release suggests the involvement of alpha3beta4(*) and at least one other nAChR subtype, thus distinguishing it from that of nAChR-evoked hippocampal [(3)H]NE release.     

Alpha-conotoxin Arenatus IB[V11L,V16D] [corrected] is a potent and selective antagonist at rat and human native alpha7 nicotinic acetylcholine receptors

A recently developed alpha-conotoxin, alpha-conotoxin Arenatus IB-[V11L,V16D] (alpha-CtxArIB[V11L,V16D]) [corrected], is a potent and selective competitive antagonist at rat recombinant alpha7 nicotinic acetylcholine receptors (nAChRs), making it an attractive probe for this receptor subtype. alpha7 nAChRs are potential therapeutic targets that are widely expressed in both neuronal and non-neuronal tissues, where they are implicated in a variety of functions. In this study, we evaluate this toxin at rat and human native nAChRs. Functional alpha7 nAChR responses were evoked by choline plus the allosteric potentiator PNU-120596 [1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea] in rat PC12 cells and human SH-SY5Y cells loaded with calcium indicators. alpha-CtxArIB[V11L,V16D] specifically inhibited alpha7 nAChR-mediated increases in Ca2+ in PC12 cells. Responses to other stimuli, 5-I-A-85380 [5-iodo-3-(2(S)-azetidinylmethoxy)pyridine dihydrochloride], nicotine, or KCl, that did not activate alpha7 nAChRs were unaffected. Human alpha7 nAChRs were also sensitive to alpha-CtxArIB[V11L, V16D]; acetylcholine-evoked currents in Xenopus laevis oocytes expressing human alpha7 nAChRs were inhibited by alpha-CtxArIB[V11L,V16D] (IC(50), 3.4 nM) in a slowly reversible manner, with full recovery taking 15 min. This is consistent with the time course of recovery from blockade of rat alpha7 nAChRs in PC12 cells. alpha-CtxArIB[V11L,V16D] inhibited human native alpha7 nAChRs in SHSY5Y cells, activated by either choline or AR-R17779 [(2)-spiro[1-azabicyclo[2.2.2]octane-3,59-oxazolidin]-29-one] plus PNU-120596. Rat brain alpha7 nAChRs contribute to dopamine release from striatal minces; alpha-CtxArIB[V11L,V16D] (300 nM) selectively inhibited choline-evoked dopamine release without affecting responses evoked by nicotine that activates heteromeric nAChRs. This study establishes that alpha-CtxArIB[V11L,V16D] selectively inhibits human and rat native alpha7 nAChRs with comparable potency, making this a potentially useful antagonist for investigating alpha7 nAChR functions.     

Melatonin - the key to the gate of sleep

This article reviews the evidence that melatonin, a hormone produced by the pineal gland during the dark hours, plays a major role in the regulation of the sleep-wake cycle. In recent years, our laboratory has been involved in a large-scale project aimed at investigating the role of endogenous melatonin in sleep-wake regulation and the effects of nonpharmacological levels of melatonin on sleep. Based on our finding on the precise coupling between the endogenous nocturnal increase in melatonin secretion and the opening of the nocturnal sleep gate, we propose that the role of melatonin in the induction of sleep does not involve the active induction of sleep, but is rather mediated by an inhibition of a wakefulness-producing mechanism in the central nervous system. Our studies also suggest that exogenously administered melatonin may be beneficial in certain types of insomnia that are related to disturbances in the normal secretion of the hormone.     

Diurnal variation of the rat vas deferens contraction induced by stimulation of presynaptic nicotinic receptors and pineal function.

A rhythmic variation of maximal contraction induced by acetylcholine in the prostatic portion of rat vas deferens was tested. This contraction is due to the release of norepinephrine and ATP from sympathetic nerve terminals. Male Wistar rats (4 months old) were housed on a light/dark cycle (12 hr/12 hr, lights on at 6:00 A.M.). The diurnal variation of acetylcholine-induced contraction was determined on animals sacrificed every 3 hr during the day. The maximal contractile response shows a circadian (24:00 hr) and an ultradian (12:20 hr) rhythm. Otherwise, the sensitivity to acetylcholine (pD2 values) and the maximal contraction or pD2 values to norepinephrine, ATP and K+ did not change throughout the day. The blocking effect of hexamethonium on the contraction induced by field stimulation was higher at 9:00 P.M. than at 3:00 P.M., indicating a diurnal variation of the effect of endogenous released acetylcholine. When melatonin released by the pineal gland is suppressed by constant illumination or superior cervical ganglionectomy, the circadian rhythm was abolished and the period of the ultradian rhythm changed to 6:30 hr. The acetylcholine-induced contraction of vasa deferentia from animals sacrificed at 3:00 P.M. and incubated "in vitro" with melatonin (100 pg/ml) increased reaching nocturnal values. In conclusion, it seems that a functional pineal gland, most probably through the synthesis and release of melatonin, is necessary for expression (circadian) and modulation (ultradian) of the rhythmicity in the maximal acetylcholine-induced contraction in the prostatic portion of the rat vas deferens.     

Interactions of MK-801 with glutamate-, glutamine- and methamphetamine-evoked release of [3H]dopamine from striatal slices.

The interactions of MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine], glutamate and glutamine with methamphetamine (METH)-evoked release of [3H]dopamine were assessed in vitro to determine whether MK-801 inhibition of METH neurotoxicity might be mediated presynaptically, and to evaluate the effects of glutamatergic stimulation on METH-evoked dopamine release. MK-801 inhibition of glutamate- or METH-evoked dopamine release might reduce synaptic dopamine levels during METH exposure and decrease the formation of 6-hydroxydopamine or other related neurotoxins. Without Mg++ present, 40 microM and 1 mM glutamate evoked a N-methyl-D-aspartate receptor-mediated [3H]dopamine and [3H]metabolite (tritium) release of 3 to 6 and 12 to 16% of total tritium stores, respectively, from striatal slices. With 1.50 mM Mg++ present, 10 mM glutamate alone or in combination with the dopamine uptake blocker nomifensine released only 2.1 or 4.2%, respectively, of total tritium stores, and release was only partially dependent on N-methyl-D-aspartate-type glutamate receptors. With or without 1.50 mM Mg++ present, 0.5 or 5 microM METH evoked a substantial release of tritium (5-8 or 12-21% of total stores, respectively). METH-evoked dopamine release was not affected by 5 microM MK-801 but METH-evoked release was additive with glutamate-evoked release. Without Mg++ present, 1 mM glutamine increased glutamate release and induced the release of [3H]dopamine and metabolites. Both 0.5 and 5 microM METH also increased tritium release with 1 mM glutamine present. When striatal slices were exposed to 5 microM METH this glutamine-evoked release of glutamate was increased more than 50%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alpha-2 adrenergic activity of bromocriptine and quinpirole in chicken pineal gland. Effects on melatonin synthesis and [3H]rauwolscine binding

In the pineal gland and retina of chickens, serotonin N-acetyl-transferase (NAT) activity and melatonin content are modulated by different receptors, alpha-2 adrenergic receptors in pineal gland and D2-dopamine receptors in retina. The effect of two D2-dopamine receptor agonists, bromocriptine and quinpirole (LY 171555), on melatonin synthesis in these tissues was investigated. Systemic administrations of bromocriptine and quinpirole decreased nocturnal NAT activity and melatonin content of both pineal gland and retina. Bromocriptine was equipotent in the two tissues, whereas quinpirole was approximately 100-fold more potent in retina than in pineal gland. In pineal gland, the suppressive effects of bromocriptine and quinpirole on NAT activity were blocked by yohimbine, a selective alpha-2 adrenergic receptor antagonist, but not by spiperone, a D2-dopamine receptor antagonist. In contrast, bromocriptine- and quinpirole-induced decreases of the enzyme activity in retina were antagonized by spiperone, and not affected by yohimbine. The nocturnal increase of NAT activity of pineal glands in vitro was inhibited with an order of potency clonidine greater than bromocriptine greater than quinpirole. Additionally, bromocriptine and quinpirole displaced the specific binding of [3H]rauwolscine, an alpha-2 adrenergic receptor antagonist, to membranes from chicken pineal gland, with potencies comparable to those observed for inhibition of NAT activity in vitro. It is suggested that bromocriptine and quinpirole, in addition to their D2-dopaminergic activity, can stimulate alpha-2 adrenergic receptors in pineal gland of chicken.     

Selective depression by general anesthetics of glutamate versus GABA release from isolated cortical nerve terminals

The role of presynaptic mechanisms in general anesthetic depression of excitatory glutamatergic neurotransmission and facilitation of GABA-mediated inhibitory neurotransmission is unclear. A dual isotope method allowed simultaneous comparisons of the effects of a representative volatile (isoflurane) and intravenous (propofol) anesthetic on the release of glutamate and GABA from isolated rat cerebrocortical nerve terminals (synaptosomes). Synaptosomes were prelabeled with L-[(3)H]glutamate and [(14)C]GABA, and release was determined by superfusion with pulses of 30 mM K(+) or 1 mM 4-aminopyridine (4AP) in the absence or presence of 1.9 mM free Ca(2+). Isoflurane maximally inhibited Ca(2+)-dependent 4AP-evoked L-[(3)H]glutamate release (99 +/- 8% inhibition) to a greater extent than [(14)C]GABA release (74 +/- 6% inhibition; P = 0.023). Greater inhibition of L-[(3)H]glutamate versus [(14)C]GABA release was also observed for the Na(+) channel antagonists tetrodotoxin (99 +/- 4 versus 63 +/- 5% inhibition; P < 0.001) and riluzole (84 +/- 5 versus 52 +/- 12% inhibition; P = 0.041). Propofol did not differ in its maximum inhibition of Ca(2+)-dependent 4AP-evoked L-[(3)H]glutamate release (76 +/- 12% inhibition) compared with [(14)C]GABA (84 +/- 31% inhibition; P = 0.99) release. Neither isoflurane (1 mM) nor propofol (15 microM) affected K(+)-evoked release, consistent with a molecular target upstream of the synaptic vesicle exocytotic machinery or voltage-gated Ca(2+) channels coupled to transmitter release. These findings support selective presynaptic depression of excitatory versus inhibitory neurotransmission by clinical concentrations of isoflurane, probably as a result of Na(+) channel blockade.