N-n-alkylnicotinium analogs,a novel class of nicotinic receptor antagonist: inhibition of S(-)-nicotine-evoked [(3)H]dopamine overflow from superfused rat striatal slices

The structure of the S(-)-nicotine molecule was modified via N-n-alkylation of the pyridine-N atom to afford a series of N-n-alkylnicotinium iodide salts with carbon chain lengths varying between C(1) and C(12). The ability of these analogs to evoke [(3)H] overflow and inhibit S(-)-nicotine-evoked [(3)H] overflow from [(3)H]dopamine ([(3)H]DA)-preloaded rat striatal slices was determined. At high concentrations, analogs with chain lengths > or =C(6) evoked [(3)H] overflow. Specifically, N-n-decylnicotinium iodide (NDNI; C(10)) evoked significant [(3)H] overflow at 1 microM, and N-n-dodecylnicotinium iodide (NDDNI; C(12)) at 10 microM, whereas N-n-octylnicotinium iodide (NONI; C(8)), N-n-heptylnicotinium iodide (NHpNI; C(7)), and N-n-hexylnicotinium iodide (C(6)) evoked [(3)H] overflow at 100 microM. Thus, intrinsic activity at these concentrations prohibited assessment of inhibitory activity. The most potent N-n-alkylnicotinium analog to inhibit S(-)-nicotine-evoked [(3)H] overflow was NDDNI, with an IC(50) value of 9 nM. NHpNI, NONI, and N-n-nonylnicotinium iodide (C(9)) also inhibited S(-)-nicotine-evoked [(3)H] overflow with IC(50) values of 0.80, 0.62, and 0.21 microM, respectively. In comparison, the competitive neuronal nicotinic acetylcholine receptor (nAChR) antagonist, dihydro-beta-erythroidine, had an IC(50) of 1.6 microM. A significant correlation of N-n-alkyl chain length with analog-induced inhibition was observed, with the exception of NDNI, which was devoid of inhibitory activity. The mechanism of N-n-alkylnicotinium-induced inhibition of the high-affinity, low-capacity component of S(-)-nicotine-evoked [(3)H] overflow was determined via Schild analysis, using the representative analog, NONI. Linear Schild regression and slope not different from unity suggested that NONI competitively interacts with a single nAChR subtype to inhibit S(-)-nicotine-evoked [(3)H]DA release (K(i) value = 80.2 nM). Thus, modification of the S(-)-nicotine molecule converts this agonist into an antagonist at nAChRs, mediating S(-)-nicotine-evoked DA release in striatum.     

N-n-alkylnicotinium analogs,a novel class of nicotinic receptor antagonists: interaction with alpha4beta2* and alpha7* neuronal nicotinic receptors

The current study demonstrates that N-n-alkylnicotinium analogs with increasing n-alkyl chain lengths from 1 to 12 carbons have varying affinity (Ki = 90 nM-20 microM) for S-(-)-[3H]nicotine binding sites in rat striatal membranes. A linear relationship was observed such that increasing n-alkyl chain length provided increased affinity for the alpha4beta2* nicotinic acetylcholine receptor (nAChR) subtype, with the exception of N-n-octylnicotinium iodide (NONI). The most potent analog was N-n-decylnicotinium iodide (NDNI; Ki = 90 nM). In contrast, none of the analogs in this series exhibited high affinity for the [3H]methyllycaconitine binding site, thus indicating low affinity for the alpha7* nAChR. The C8 analog, NONI, had low affinity for S-(-)-[3H]nicotine binding sites but was a potent inhibitor of S-(-)-nicotine-evoked [3H]dopamine (DA) overflow from superfused striatal slices (IC50 = 0.62 microM), thereby demonstrating selectivity for the nAChR subtype mediating S-(-)-nicotine-evoked [3H]DA overflow (alpha3alpha6beta2* nAChRs). Importantly, the N-n-alkylnicotinium analog with highest affinity for the alpha4beta2* subtype, NDNI, lacked the ability to inhibit S-(-)-nicotine-evoked [3H]DA overflow and, thus, appears to be selective for alpha4beta2* nAChRs. Furthermore, the present study demonstrates that the interaction of these analogs with the alpha4beta2* subtype is via a competitive mechanism. Thus, selectivity for the alpha4beta2* subtype combined with competitive interaction with the S-(-)-nicotine binding site indicates that NDNI is an excellent candidate for studying the structural topography of alpha4beta2* agonist recognition binding sites, for identifying the antagonist pharmacophore on the alpha4beta2* nAChR, and for defining the role of this subtype in physiological function and pathological disease states.     

The selective serotonin reuptake inhibitor citalopram induces the storage of serotonin in catecholaminergic terminals

We investigated whether selective inhibition of serotonin (5-hydroxytryptamine; 5-HT) transporter with citalopram leads to accumulation of 5-HT in catecholaminergic neurons. In the rabbit olfactory tubercle, citalopram (1-10 microM) inhibited [(3)H]5-HT uptake; however, the maximal degree of inhibition achieved was 70%. Addition of nomifensine (1-10 microM) was required for complete inhibition of [(3)H]5-HT uptake. In slices labeled with 0.1 microM [(3)H]5-HT, cold 5-HT (0.03-1 microM) induced a large increase in the efflux (release) of stored [(3)H]5-HT, an effect blocked by coperfusion with 1 microM citalopram. Similar concentrations (0.03-1 microM) of norepinephrine (NE) or dopamine (DA) failed to release [(3)H]5-HT. When labeling with 0.1 microM [(3)H]5-HT was carried out in the presence of citalopram, 1) low concentrations of 5-HT failed to release [(3)H]5-HT; 2) DA and NE were more potent and effective in releasing [(3)H]5-HT than in control slices; 3) coperfusion of NE, DA, or 5-HT with citalopram enhanced the release of [(3)H]5-HT induced by the catecholamines but not by 5-HT; and 4) coperfusion of NE or DA with nomifensine antagonized NE- and DA-evoked [(3)H]5-HT release, with a greater effect on NE than on DA. These results suggest that in the rabbit olfactory tubercle, where there is coexistence of 5-HT, NE, and DA neurons, inhibition of the 5-HT transporter led to accumulation of 5-HT in catecholaminergic terminals. Thus, during treatment with selective serotonin uptake inhibitors (SSRIs), 5-HT may be stored in catecholaminergic neurons acting as a false neurotransmitter and/or affecting the disposition of DA and/or NE. Transmitter relocation may be involved in the antidepressant action of SSRIs.     

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.     

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.     

Multiple cellular mechanisms mediate the effect of lobeline on the release of norepinephrine

The complex effect of lobeline on [(3)H]norepinephrine ([(3)H]NE) release was investigated in this study. Lobeline-induced release of [(3)H]NE from the vas deferens was strictly concentration-dependent. In contrast, electrical stimulation-evoked release was characterized by diverse effects of lobeline depending on the concentration used: at lower concentration (10 microM), it increased the release and at high concentration (100 and 300 microM), the evoked release of [(3)H]NE was abolished. The effect of lobeline on the basal release was [Ca(2+)]-independent, insensitive to mecamylamine, a nicotinic acetylcholine receptor antagonist, and to desipramine, a noradrenaline uptake inhibitor. However, lobeline-induced release was temperature-dependent: at low temperature (12 degrees C), at which the membrane carrier proteins are inhibited, lobeline failed to increase the basal release. Lobeline dose dependently inhibited the uptake of [(3)H]NE into rat hippocampal synaptic vesicles and purified synaptosomes with IC(50) values of 1.19 +/- 0.11 and 6.53 +/- 1.37 microM, respectively. Lobeline also inhibited Ca(2+) influx induced by KCl depolarization in sympathetic neurons measured with the Fura-2 technique. In addition, phenylephrine, an alpha(1)-adrenoceptor agonist, contracted the smooth muscle of the vas deferens and enhanced stimulation-evoked contraction. Both effects were inhibited by lobeline. Our results can be best explained as a reversal of the monoamine uptake by lobeline that is facilitated by the increased intracellular NE level after lobeline blocks vesicular uptake. At high concentrations, lobeline acts as a nonselective Ca(2+) channel antagonist blocking pre- and postjunctional Ca(2+) channels serving as a counterbalance for the multiple transmitter releasing actions.     

Analgesic and toxic effects of ABT-594 resemble epibatidine and nicotine in rats

The present study directly compared the antinociceptive and toxic effects of the neuronal nicotinic receptor agonist ABT-594 ((R)-5-(2-azetidinylmethoxy)-2-chloropyridine) with (-)-nicotine and (+)-epibatidine. Like (-)-nicotine (0.8 and 1.6 mg/kg s.c.) and (+)-epibatidine (0.005 and 0.01 mg/kg s.c.), ABT-594 (0.05 and 0.1 mg/kg s.c.) increased response latencies in the hot-plate test in rats, indicating that it has antinociceptive activity. In contrast to (-)-nicotine and (+)-epibatidine, ABT-594 did not cause rotarod impairment at antinociceptive doses but did cause hypothermia and life-threatening adverse effects including seizures. ABT-594 (0.01 and 0.1 mg/kg i.v.) also produced a dose-dependent increase in blood pressure resembling that observed with (-)-nicotine (0.03, 0.1 and 0. 03 mg/kg i.v.) and (+)-epibatidine (0.001 and 0.003 mg/kg i.v.). Both the antinociceptive and toxic effects (convulsions and hypertension) were abolished by pretreatment with the brain penetrant neuronal nAChR antagonist mecamylamine (1 mg/kg s.c.; i.v. for cardiovascular studies), demonstrating that these actions of ABT-594 were mediated via activation of neuronal nicotinic receptors. Continuous infusion of ABT-594 (0.2 mg/kg per day s.c.) to rats for 7 days followed by challenge with mecamylamine (1 mg/kg i.p.) induced a nicotine-like abstinence syndrome suggesting that ABT-594 has nicotine-like dependence liability. These findings indicate that the acute safety profile of ABT-594 is not significantly improved over other nicotinic analgesics.     

LLC-PK(1) cells stably expressing the human norepinephrine transporter: A functional model of carrier-mediated norepinephrine release in protracted myocardial ischemia.

In myocardial ischemia, adrenergic terminals undergo ATP depletion, hypoxia, and intracellular pH reduction, causing the accumulation of axoplasmic norepinephrine (NE) and intracellular Na(+) [via the Na(+)-H(+) exchanger (NHE)]. This forces the reversal of the Na(+)- and Cl(-)-dependent NE transporter (NET), triggering massive carrier-mediated NE release and, thus, arrhythmias. We have now developed a cellular model of carrier-mediated NE release using an LLC-PK(1) cell line stably transfected with human NET cDNA (LLC-NET). LLC-NET cells transported [(3)H]NE and [(3)H]N-methyl-4-phenylpyridinium ([(3)H]MPP(+)) in an inward direction. This uptake was abolished by the NET inhibitors desipramine (100 nM) and mazindol (300 nM) and by extracellular Na(+) removal. Na(+)-gradient reversal induced an efflux of (3)H-substrate from preloaded LLC-NET cells. Desipramine and mazindol blocked this efflux. Because of its greater intracellular stability and higher sensitivity to Na(+)-gradient reversal, [(3)H]MPP(+) proved preferable to [(3)H]NE as an NET substrate; therefore, only [(3)H]MPP(+) was used for subsequent studies. The K(+)/H(+) ionophore nigericin (10 microM) evoked a large efflux of [(3)H]MPP(+). This efflux was potentiated by the Na(+),K(+)-ATPase inhibitor ouabain (100 microM), was sensitive to desipramine, and was blocked by the NHE inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA; 10 microM). In contrast, EIPA failed to inhibit the [(3)H]MPP(+) efflux elicited by the Na(+) ionophore gramicidin (10 microM). Furthermore, [(3)H]MPP(+) efflux induced by the NHE-stimulant proprionate (25 mM) was negatively modulated by imidazoline receptor activation. Our findings suggest that LLC-NET cells are a sensitive model for studying transductional processes of carrier-mediated NE release associated with myocardial ischemia.     

Stimulus-dependent modulation of [(3)H]norepinephrine release from rat neocortical slices by gabapentin and pregabalin

Gabapentin (GBP; Neurontin) has proven efficacy in several neurological and psychiatric disorders yet its mechanism of action remains elusive. This drug, and the related compounds pregabalin [PGB; CI-1008, S-(+)-3-isobutylgaba] and its enantiomer R-(-)-3-isobutylgaba, were tested in an in vitro superfusion model of stimulation-evoked neurotransmitter release using rat neocortical slices prelabeled with [(3)H]norepinephrine ([(3)H]NE). The variables addressed were stimulus type (i.e., electrical, K(+), veratridine) and intensity, concentration dependence, onset and reversibility of action, and commonality of mechanism. Both GBP and PGB inhibited electrically and K(+)-evoked [(3)H]NE release, but not that induced by veratridine. Inhibition by these drugs was most pronounced with the K(+) stimulus, allowing determination of concentration-effect relationships (viz., 25 mM K(+) stimulus: GBP IC(50) = 8.9 microM, PGB IC(50) = 11.8 microM). R-(-)-3-Isobutylgaba was less effective than PGB to decrease stimulation-evoked [(3)H]NE release. Other experiments with GBP demonstrated the dependence of [(3)H]NE release inhibition on optimal stimulus intensity. The inhibitory effect of GBP increased with longer slice exposure time before stimulation, and reversed upon washout. Combination experiments with GBP and PGB indicated a similar mechanism of action to inhibit K(+)-evoked [(3)H]NE release. GBP and PGB are concluded to act in a comparable, if not identical, manner to preferentially attenuate [(3)H]NE release evoked by stimuli effecting mild and prolonged depolarizations. This type of modulation of neurotransmitter release may be integral to the clinical pharmacology of these drugs.     

Changes in sensitivity of release modulating dopamine autoreceptors after chronic treatment with haloperidol

The release of recently taken up [3H]dopamine ([3H]DA) elicited by electrical stimulation (3 Hz, 2 min, 16 mA) from slices of the rabbit caudate nucleus is inhibited by apomorphine (0.01-0.1 microM) in a concentration-dependent manner. This action is mediated through the activation of presynaptic inhibitory DA autoreceptors. The inhibition of [3H]DA release by apomorphine (0.1 microM) was antagonized 2 hr, but not 24 hr after the single administration of haloperidol (1 mg/kg s.c.). After 2 days of withdrawal after 28 days of chronic treatment with haloperidol (1 mg/kg s.c.) once daily, apomorphine (0.01-0.1 microM) was more effective in inhibiting [3H]DA release elicited by electrical stimulation when compared with rabbits injected chronically with either the vehicle for haloperidol or with saline. In superfused slices of the rabbit caudate nucleus, exposure to S-sulpiride (0.1 and 1 microM) increased in a concentration-dependent manner the release of [3H] DA elicited by electrical stimulation. After 28 days of chronic treatment with haloperidol, the facilitation of [3H]DA release by S-sulpiride was significantly reduced when compared with the controls. The inhibition of central noradrenergic transmission by DA receptor agonists was studied in hypothalamic slices prelabeled with [3H]norepinephrine ([3H-NE]). Apomorphine (0.01-1 microM) inhibited the electrically evoked (5 Hz, 2 min, 26 mA) release of [3H]NE from hypothalamic slices of untreated rabbits. The sensitivity to the inhibitory effect of apomorphine on [3H]NE overflow remained unaffected after 2 days of withdrawal following 28 days of chronic treatment with haloperidol. In summary, our results indicate that chronic haloperidol administration induces changes in sensitivity of the DA autoreceptors regulating dopaminergic neurotransmission but does not affect the sensitivity of DA receptors modulating NE release in the central nervous system. These results suggest that the DA autoreceptors that regulate dopaminergic neurotransmission may play a physiological role in the modulation of transmitter release and consequently are susceptible to the development of changes in sensitivity after chronic receptor blockade. The possible implication of changes in sensitivity of the DA autoreceptor during the treatment of schizophrenia with neuroleptics is discussed.     

(S)-(-)-Cotinine, the major brain metabolite of nicotine, stimulates nicotinic receptors to evoke [3H]dopamine release from rat striatal slices in a calcium-dependent manner

Cotinine, a major peripheral metabolite of nicotine, has recently been shown to be the most abundant metabolite in rat brain after peripheral nicotine administration. However, little attention has been focused on the contribution of cotinine to the pharmacological effects of nicotine exposure in either animals or humans. The present study determined the concentration-response relationship for (S)-(-)-cotinine-evoked 3H overflow from superfused rat striatal slices preloaded with [3H]dopamine ([3H]DA) and whether this response was mediated by nicotinic receptor stimulation. (S)-(-)-Cotinine (1 microM to 3 mM) evoked 3H overflow from [3H]DA-preloaded rat striatal slices in a concentration-dependent manner with an EC50 value of 30 microM, indicating a lower potency than either (S)-(-)-nicotine or the active nicotine metabolite, (S)-(-)-nornicotine. As reported for (S)-(-)-nicotine and (S)-(-)-nornicotine, desensitization to the effect of (S)-(-)-cotinine was observed. The classic nicotinic receptor antagonists mecamylamine and dihydro-beta-erythroidine inhibited the response to (S)-(-)-cotinine (1-100 microM). Additionally, 3H overflow evoked by (S)-(-)-cotinine (10-1000 microM) was inhibited by superfusion with a low calcium buffer. Interestingly, over the same concentration range, (S)-(-)-cotinine did not inhibit [3H]DA uptake into striatal synaptosomes. These results demonstrate that (S)-(-)-cotinine, a constituent of tobacco products and the major metabolite of nicotine, stimulates nicotinic receptors to evoke the release of DA in a calcium-dependent manner from superfused rat striatal slices. Thus, (S)-(-)-cotinine likely contributes to the neuropharmacological effects of nicotine and tobacco use.     

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.     

(+)-Fenfluramine and its major metabolite, (+)-norfenfluramine,are potent substrates for norepinephrine transporters

(+/-)-Fenfluramine is an amphetamine analog that was once widely prescribed as an appetite suppressant. Although (+/-)-fenfluramine is no longer clinically available, the mechanisms underlying its anorectic properties are still of interest. Upon peripheral administration, stereoisomers of (+/-)-fenfluramine are N-deethylated to form the metabolites, (+)- and (-)-norfenfluramine. It is well accepted that isomers of (+/-)-fenfluramine and (+/-)-norfenfluramine interact with 5-hydroxytryptamine (serotonin, 5-HT) transporters to release 5-HT from neurons. However, the effects of these drugs on other monoamine transporters are not well characterized. In this study, we examined the interaction of stereoisomers of (+/-)-fenfluramine and (+/-)-norfenfluramine with transporters for 5-HT, norepinephrine (NE), and dopamine (DA). Results from in vitro assays confirmed these drugs are potent substrates for 5-HT transporters: (+)-fenfluramine, (-)-fenfluramine, (+)-norfenfluramine, and (-)-norfenfluramine released [3H]5-HT from synaptosomes with EC50 values of 52, 147, 59, and 287 nM, respectively. Importantly, (+)-fenfluramine and (+)-norfenfluramine released [3H]NE with EC50 values of 302 and 73 nM. Results from in vivo microdialysis experiments showed that intravenous injection of (+)-norfenfluramine elevates extracellular levels of 5-HT, NE, and DA in rat frontal cortex. The effects of (+)-norfenfluramine on NE and DA were antagonized by pretreatment with the NE uptake blocker nisoxetine. In summary, administration of fenfluramines can increase synaptic levels of 5-HT, NE, and DA in the cortex, and (+)-norfenfluramine likely contributes to these effects. Release of NE and DA evoked by (+)-norfenfluramine is at least partly mediated via NE transporters. Our results emphasize the potential involvement of noradrenergic mechanisms in the actions of fenfluramines.     

Ethanol inhibits N-methyl-D-aspartate-stimulated [3H]norepinephrine release from rat cortical slices

The effects of ethanol on N-methyl-D-aspartate (NMDA)-stimulated [3H]norepinephrine (NE) release from rat cortical slices was studied. NMDA-stimulated [3H]NE release was inhibited by tetrodotoxin, Mg++ and 2-amino-5-phosphonopentanoic acid, indicating that NMDA receptors in the cortex have characteristics similar to those observed using electrophysiological studies. Ethanol (60-200 mM) decreased the release of [3H]NE evoked by 100 microM NMDA in a concentration-dependent manner (32-52% inhibition), but it did not significantly alter the basal release. The inhibitory effect of 100 mM ethanol was due to a reduction in the maximal response with no significant change in the EC50 for NMDA. Pretreatment of the slices with 100 mM ethanol up to 6 min did not alter the magnitude of inhibition. The inhibition of NMDA-stimulated [3H]NE release due to ethanol was reversible after a 13-min recovery period. The presence of ethanol did not significantly affect the IC50 for Mg++ inhibition of NMDA-stimulated [3H]NE release (23 +/- 3 microM). Glycine (10-300 microM) potentiated the release of [3H]NE stimulated by 250 microM NMDA, and 60 mM ethanol did not alter this effect of glycine. Ethanol (100 mM) inhibited the release of [3H]NE evoked by 18.9 mM KCl in the presence or absence of 2-amino-5-phosphonopentanoic acid, but had no effect on release induced by 49.1 mM KCl. Tetrodotoxin (0.3 mM) significantly decreased the release of [3H] NE evoked by 23.2 mM KCl, and 60 to 200 mM ethanol did not alter this release. These results suggest that NMDA receptors in rat cortical slices are located on nerve cell bodies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction between serotonin uptake inhibitors and alpha-2 adrenergic heteroreceptors in the rat hypothalamus

The effectiveness of presynaptic receptor agonists to inhibit the electrically evoked release of [3H]monoamines from brain slices is attenuated in the presence of blockade of neuronal uptake for the serotonin (5-HT) and the norepinephrine (NE) systems. There is controversy, however, as to the existence of a functional link between the presynaptic receptors and the neuronal uptake carriers. An alternative hypothesis involves competition for the presynaptic receptor sites between the exogenous agonist and the released neurotransmitter. In order to examine the proposed functional interaction, we studied the alpha-2 adrenoceptor-mediated inhibition of the electrically evoked release of [3H]-5-HT from slices of the rat hypothalamus, a model in which endogenous NE does not activate the alpha-2 heteroreceptors located on 5-HT terminals. The inhibitors of 5-HT uptake, citalopram (0.01-1 microM) and paroxetine (1 microM), which by themselves did not modify [3H]-5-HT release, antagonized the inhibition of [3H]-5-HT overflow produced by UK 14.304, an alpha-2 adrenoceptor agonist. The inhibition of the electrically evoked release of [3H]-5-HT by exogenous NE (0.1-1 microM) was also attenuated in the presence of citalopram. In contrast, citalopram did not modify the electrically evoked release of [3H]-NE or the inhibition of [3H]-NE release mediated by UK 14.304. When the 5-HT autoreceptor was blocked by cyanopindolol, the inhibitory effect of UK 14.304 on [3H]-5-HT release was unaltered in the presence of citalopram.(ABSTRACT TRUNCATED AT 250 WORDS)     

N-methyl-D-aspartic acid (NMDA) and non-NMDA receptors regulating hippocampal norepinephrine release. II. Evidence for functional cooperation and for coexistence on the same axon terminal.

The possible interactions between activation of N-methyl-D-aspartic acid (NMDA) receptors and non-NMDA receptors regulating the release of [3H]norepinephrine [( 3H]NE) have been investigated in superfused synaptosomes from rat hippocampus. NMDA--at a concentration (100 microM) which, in a medium containing 1.2 mM Mg++ ions, did not evoke [3H]NE release--acquired releasing activity in the presence of equimolar concentrations of quisqualic acid (QA), (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or kainic acid. The [3H] NE release evoked by NMDA plus QA in the presence of Mg++ ions was Ca(++)-dependent, partly tetrodotoxin-sensitive, inhibited by clonidine but insensitive to desipramine. The NMDA receptor antagonists D-2-amino-5-phosphonopentanoic acid (D-AP5) and (+)-5-methyl-10,11-dihydro-5-H-dibenzo[a,d]cycloepten-5,10-imine (MK-801) antagonized the NMDA-induced [3H]NE release in Mg(++)-free medium; the IC50 values amounted, respectively, to 81.4 microM and to 1.11 microM. When NMDA was tested in the presence of QA and Mg++ ions, the affinity of D-AP5 was enormously increased (IC50 = 40 nM; i.e., more than 6 orders of magnitude); the affinity of MK-801 was found to be augmented by 350-fold.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of amiloride and 5-(N,N-hexamethylene)amiloride on dopaminergic and cholinergic neurotransmission

In order to establish the role of the Na+/H+ exchange transport on neurotransmission, we investigated the effects of amiloride and of 5-(N,N-hexamethylene)amiloride (HMA) on dopamine (DA) and acetylcholine (ACh) release and on receptor-mediated modulation of DA and ACh release. Superfused rabbit striatal slices prelabeled with [3H]DA and [14C]choline were stimulated electrically in the presence and absence of several concentrations of these agents. Amiloride (3-10 microM) and HMA (0.3-10 microM) reduced the basal efflux and the stimulation evoked overflow of total 3H and of [3H]-3,4-dihydroxyphenylacetic acid and inhibited monoamine oxidase activity. The inhibition of stimulation evoked overflow of total 3H was blocked by pretreatment with nomifensine but not by sulpiride. Amiloride had no effect on the basal efflux and the stimulation evoked overflow of ACh or it did modify apomorphine-induced inhibition of DA and ACh release. However, at 3 to 10 microM, HMA enhanced the basal efflux of 3H; this effect was not prevented either by uptake inhibition with nomifensine or by low extracellular calcium. These results suggest that amiloride-sensitive Na+ transport and the amiloride and HMA-sensitive Na+/H+ antiporter play no role on the secretion of DA and ACh, or on the mechanisms by which activation of pre- and postsynaptic DA receptors lead to inhibition of neurotransmitter release. Amiloride- and HMA-induced monoamine oxidase inhibition accounts for the effects of amiloride and HMA on DA efflux and overflow. The guanidine moiety present in the amiloride and HMA molecules is most likely responsible for these effects.     

Long-lasting facilitation of 4-amino-n-[2,3-(3)H]butyric acid ([(3)H]GABA) release from rat hippocampal slices by nicotinic receptor activation

In this study we explored the effect of the stimulation of nicotinic acetylcholine receptors located on interneurons by measuring 4-amino-n-[2,3-(3)H]butyric acid ([(3)H]GABA) release and monitoring [Ca (2+)](i) in superfused hippocampal slices. In the presence of 6-cyano-7-nitroquinoxaline-2,3-dione, (+/-)-2-amino-5-phosphonopentanoic acid, and atropine, i.e., under the blockade of N-methyl-D-aspartate and non-N-methyl-D-aspartate glutamate and muscarinic receptors, nicotine did not alter the spontaneous outflow of [(3)H]GABA, but significantly increased the stimulation-evoked [(3)H]GABA efflux. This effect of nicotine depended on the time interval between nicotine treatment and electrical stimulus, the concentration of nicotine (1-100 microM), and the parameters of electrical depolarization. Acetylcholine (0.03-3 mM), and the alpha 7 subtype-selective agonist choline (0.1-10 mM), also potentiated stimulus-evoked release of [(3)H]GABA, whereas 1,1-dimethyl-4-phenilpiperazinium iodide failed to increase the tritium outflow significantly. The effect of nicotine treatment was prevented by tetrodotoxin (1 microM) and by the nicotinic acetylcholine receptor antagonist mecamylamine (10 microM), and the alpha 7 subtype-selective antagonists alpha-bungarotoxin (100 nM) and methyllycaconitine (10 nM), whereas dihidro-beta-erythroidine (20 nM) was without effect. Perfusion of 100 microM nicotine caused a [Ca(2+)](i) transient in about one-third of the tested interneurons; however, the response to subsequent electrical stimulation remained unchanged. Inhibition of the GABA transporter system by nipecotic acid (1 mM) or by decreasing the bath temperature to 12 degrees C abolished completely the effect of nicotine to potentiate the stimulation-evoked release of GABA. These findings indicate that the activation of alpha 7-type nicotinic receptors of hippocampal interneurons results in a long-lasting ability of these cells to respond to depolarization with an increased release of GABA mediated by the transporter system.     

Frequency-dependent muscarinic receptor modulation of acetylcholine and dopamine release from rabbit striatum

The effects of muscarinic receptor activation on the electrically evoked release of [3H]dopamine (DA) and [14C]acetylcholine (ACh) or [3H]ACh were investigated in rabbit striatal slices. Release was measured in the presence of 10 microM hemicholinium and 1 microM sulpiride to block choline uptake and prevent the effects of released DA on DA receptors modulating release. Stimulation (120 pulses, 20 mA, 2 msec) at 0.3, 3 and 10 Hz produced (3H or 14C) ACh release that sharply declined with increasing stimulation frequency. A flat frequency-release curve was obtained for DA. Oxotremorine (OXO), a direct muscarinic agonist (1-100 microM), produced a concentration-dependent inhibition of ACh release, inversely related to stimulation frequency, at a fixed number of pulses (120). When the number of pulses was modified to produce similar amounts of ACh release (20 pulses at 0.1 Hz, 39 pulses at 0.3 Hz, 120 pulses at 3 Hz and 350 pulses at 10 Hz), much greater inhibition of ACh release by OXO (0.3 and 3 microM) was obtained with lower frequencies and lower number of pulses. Physostigmine, an acetylcholinesterase inhibitor, decreased ACh release with an inverse relationship to stimulation frequency. Atropine (1 microM), a selective muscarinic antagonist, enhanced the release of ACh more at 10 Hz than at 0.3 and 3 Hz and completely antagonized the effects of OXO (10 microM) and physostigmine (1 microM) at all three stimulation frequencies. OXO (3 and 10 microM) enhanced DA release at 3 Hz. Physostigmine (1 microM) and atropine (1 microM) had no effect on DA release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antidepressant drug-induced alterations in neuron-localized tumor necrosis factor-alpha mRNA and alpha(2)-adrenergic receptor sensitivity

The pleiotropic cytokine tumor necrosis factor-alpha (TNF) and alpha(2)-adrenergic receptor activation regulate norepinephrine (NE) release from neurons in the central nervous system. The present study substantiates the role of TNF as a neuromodulator and demonstrates a reciprocally permissive relationship between the biological effects of TNF and alpha(2)-adrenergic receptor activation as a mechanism of action of antidepressant drugs. Immunohistochemical analysis and in situ hybridization reveal that administration of the antidepressant drug desipramine decreases the accumulation of constitutively expressed TNF mRNA in neurons of the rat brain. Superfusion and electrical field stimulation were applied to a series of rat hippocampal brain slices to study the regulation of [(3)H]NE release. Superfusion of hippocampal slices obtained from rats chronically administered the antidepressant drug zimelidine demonstrates that TNF-mediated inhibition of [(3)H]NE release is transformed, such that [(3)H]NE release is potentiated in the presence of TNF, an effect that occurs in association with alpha(2)-adrenergic receptor activation. However, chronic zimelidine administration does not alter stimulation-evoked [(3)H]NE release, whereas chronic desipramine administration increases stimulation-evoked [(3)H]NE release and concomitantly decreases alpha(2)-adrenergic autoreceptor sensitivity. Collectively, these data support the hypothesis that chronic antidepressant drug administration alters alpha(2)-adrenergic receptor-dependent regulation of NE release. Additionally, these data demonstrate that administration of dissimilar antidepressant drugs similarly transform alpha(2)-adrenergic autoreceptors that are functionally associated with the neuromodulatory effects of TNF, suggesting a possible mechanism of action of antidepressant drugs.