Effects of selected muscarinic cholinergic antagonists on [3H]acetylcholine release from rat hippocampal slices

A number of cholinergic muscarinic (M) agonists and antagonists were studied for their ability to enhance tritiated acetylcholine ([3H]ACh) release from electrically field-stimulated rat hippocampal slices. A Ca++-free medium and carbachol, but not nicotine, inhibited [3H]ACh release. Atropine, methylatropine and dexetimide produced concentration-dependent increases in [3H]ACh release to a maximum of about 50% above control. Aprophen and benactyzine produced a maximal response 25 to 35% above control. The selective M1 antagonist pirenzepine had the least effect on [3H]ACh release. Of the nonspecific M1-M2 antagonists studied, benactyzine produced the least amount of [3H]ACh release. The order of potency of the M antagonists in promoting a 15% increase in [3H]ACh release was aprophen greater than benactyzine greater than methylatropine greater than dexetimide greater than pirenzepine greater than atropine. However, the order of promoting maximal release of [3H]ACh was atropine greater than dexetimide greater than methylatropine greater than aprophen greater than benactyzine greater than pirenzepine.     

Release of gamma-[3H]aminobutyric acid (GABA) from electrically stimulated rat cortical slices and its modulation by GABAB autoreceptors

Slices of rat temporo-parietal cortex were prelabeled with gamma-[3H]aminobutyric acid ([3H]GABA), in the presence of the glial GABA uptake inhibitor beta-alanine. The slices were then superfused with a medium containing the GABA transaminase inhibitor aminooxyacetic acid and stimulated electrically (5 min, 2 msec, 36 mA at 5 or 10 Hz), in the presence of the neuronal GABA reuptake inhibitor SK&F 89976A [N-(4,4-diphenyl-3-butenyl)-nipecotic acid] and of beta-alanine. Representative experiments showed that the tritium released could be accounted for almost entirely by authentic [3H]GABA. The electrically evoked overflow of [3H]GABA was tetrodotoxin sensitive and largely calcium-dependent. Exogenous GABA, added to the superfusion medium at 3 to 30 microM, reduced in a concentration-dependent manner the electrically evoked (5 Hz) release of [3H]GABA. The GABAB receptor agonist (-)-baclofen, but not the GABAA receptor agonist muscimol, mimicked GABA and produced a concentration-inhibition curve almost superimposable to that of the natural transmitter. The effects of GABA and of (-)-baclofen were much more pronounced at 5 than at 10 Hz. The GABA-induced inhibition of [3H]GABA release was sensitive to the novel GABAB receptor antagonist beta-(p-chlorophenyl)-3-amino propyl phosphonic acid which, by itself, increased the [3H]GABA overflow. The inhibitory effect of GABA was not counteracted by the GABAA receptor antagonists bicuculline or SR 95531 [2-(3'-carbethoxy-2'-propenyl)-3-amino-6-paramethoxy-phenyl-pyr idazinium bromide]. The results are compatible with the presence in the rat cerebral cortex of autoreceptors mediating inhibition of GABA release and belonging to the GABAB type. These autoreceptors may be activated tonically under physiological conditions.     

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)     

Melatonin nocturnal surge modulates nicotinic receptors and nicotine-induced [3H]glutamate release in rat cerebellum slices

In mammals, the most important synchronizer for endogenous rhythms is the environmental light/dark cycle. In this report we have explored the ability of light/dark cycle and melatonin, the pineal hormone released during the night, to modulate cerebellar cholinergic input by interfering with the nicotinic acetylcholine receptors' (nAChRs) availability. Through the analysis of the response to selective cholinergic agonists and antagonists, we observed that nAChRs containing the alpha7 gene product mediate the release of [(3)H]glutamate from rat cerebellum slices. The [(3)H]glutamate overflow induced by alpha7 nAChR activation was higher during the dark phase, although the number of alpha-[(125)I]bungarotoxin binding sites, but not the [(3)H]nicotine binding sites (B(max)), was reduced. On the other hand, glutamate-evoked [(3)H]glutamate release was not modified by the hour of the day. Finally, we show that the nocturnal increase in nicotine-evoked [(3)H]glutamate release is imposed by a nocturnal surge of melatonin, as it is abolished when pineal melatonin production is inhibited by either maintaining the animals in constant light for 48 h or by injecting propranolol just before lights off for 2 days. The difference between light and dark [(3)H]glutamate-evoked release is restored in propranolol-treated animals that received melatonin during the dark period. In conclusion, we show that nicotine-evoked [(3)H]glutamate release in rat cerebellum presents a diurnal variation, driven by nocturnal pineal melatonin surge.     

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.     

(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.     

A comparison of N-methyl-D-aspartate-evoked release of adenosine and [3H]norepinephrine from rat cortical slices

Tetrodotoxin reduced N-methyl-D-aspartate (NMDA)-evoked release of adenosine by 35% but virtually abolished [3H]norepinephrine release. Although [3H]norepinephrine release from rat cortical slices evoked by 500 microM NMDA was abolished by 1.2 mM Mg++, which produces a voltage-sensitive, uncompetitive block of NMDA-channels, adenosine release was increased in the presence of Mg++. Partial depolarization with 12 mM K+ relieved the Mg++ block of 500 microM NMDA-evoked [3H]norepinephrine release but did not affect adenosine release, indicating that a Mg++ requirement for the adenosine release process per se cannot account for this discrepancy. NMDA was 33 times more potent in releasing adenosine than [3H]norepinephrine. At submaximal concentrations of NMDA (10 and 20 microM), adenosine release was augmented in Mg+(+)-free medium. Although a high concentration of the uncompetitive NMDA antagonist MK-801 [(+)-5-methyl-10,11,dihydro-5H-dibenzo[a,d]cyclohepten-5-10-imine maleate] (3 microM) blocked NMDA-evoked release of [3H]norepinephrine and adenosine, a lower concentration (300 nM) decreased NMDA-evoked [3H]norepinephrine release by 66% without affecting adenosine release. These findings suggest that maximal adenosine release occurs when relatively few NMDA receptors are activated, raising the possibility that spare receptors exist for NMDA-evoked adenosine release. Rather than acting as a protectant against excessive NMDA excitation, released adenosine might provide an inhibitory threshold which must be overcome for NMDA-mediated neurotransmission to proceed.     

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.     

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.     

Effects of phencyclidine on the release of radioactivity from rat striatal slices labeled with [3H]choline

Phencyclidine (PCP) has been shown to antagonize the effects of acetylcholine (ACh) in a variety of systems and to inhibit the binding of muscarinic antagonists to brain membranes. Therefore, we have studied the effects of PCP on ACh release from rat striatal slices in order to characterize the effect of PCP in a central cholinergic system. After incubation with [3H]choline, striatal slices were superfused and the superfusate was assayed for radioactivity. The presence of PCP led to dose-related inhibition of K+-stimulated ACh release. This may be due to the reported ability of PCP to enhance dopamine release as both direct (pergolide) and indirect (amphetamine) dopamine agonists also inhibited striatal ACh release. Haloperidol blocked the inhibitory effects of PCP, amphetamine and pergolide on ACh release, supporting the notion that the decrease in ACh release produced by PCP is mediated indirectly via the release of dopamine onto cholinergic neurons.     

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.     

Desensitization of nicotinic agonist-induced [3H]gamma-aminobutyric acid release from mouse brain synaptosomes is produced by subactivating concentrations of agonists

Several neurochemical and electrophysiological studies have shown that neuronal nicotinic receptors are desensitized by pretreatment with lower agonist concentrations than are required to activate the receptors, but the extent of desensitization and agonist concentration required to produce desensitization vary depending upon receptor subtype. Recently, we reported that nicotinic agonists will stimulate the release of [3H]gamma-aminobutyric acid (GABA) from synaptosomes prepared from mouse brain. The studies described herein evaluated desensitization of [3H]GABA release produced by pretreatment with 12 nicotinic agonists. Pretreatment produced near total desensitization that developed slowly (onset T(1/2) = 3.46 min) and was totally reversible (recovery T(1/2) = 4.95 min). Nine of the 12 compounds tested induced total or near total desensitization at concentrations that were less than those required to produce a reliably measured increase in [3H]GABA release. Nicotine produced total block with an IC(50) value of 26 nM. This value is two orders of magnitude lower than the EC(50) for nicotine-induced [3H]GABA release (1630 nM). The three compounds that showed an overlap of the desensitization and activation concentration-effect curves (cytisine, anabasine, nornicotine) are all partial agonists. Comparison of the desensitization properties of the [3H]GABA release with an ion ((86)Rb+) efflux that we have measured previously suggests that the receptor that mediates GABA release and (86)Rb(+) efflux is the same, most likely the alpha4beta2 subtype.     

The neurotoxic compound N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP4) depletes endogenous norepinephrine and enhances release of [3H]norepinephrine from rat cortical slices

The alkylating compound N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP4) injected to rodents blocks norepinephrine (NE) uptake and reduces endogenous NE levels in the central nervous system and in the periphery. To investigate the processes leading to these alterations, rat cortical slices were incubated in the presence of DSP4. Cortical NE was depleted by 40% after incubation of slices in 10(-5) M DSP4 for 60 min and this was blocked by desipramine. The spontaneous outflow of radioactivity from cortical slices labeled previously with [3H]NE was enhanced markedly both during exposure to DSP4 and during the subsequent washings, suggesting that NE depletion could be due to this stimulation of NE release. The radioactivity released by DSP4 was accounted for mainly by NE and its deaminated metabolite 3,4-dihydroxyphenylglycol. The enhanced release, independent of external Ca++, apparently originated from the vesicular pool as it was absent after reserpine pretreatment. Activities of the enzymes related to NE synthesis were not altered by DSP4 in vitro and only monoamine oxidase activity was inhibited at high concentrations. Thus, the depletion of endogenous NE produced by DSP4 is probably due to a persistent enhancement of its release from the vesicular pool. Fixation of DSP4 to the NE transport system is necessary but not sufficient to produce the acute NE depletion and the characteristic long-term actions of the compound.     

Further characterization of the presynaptic alpha-1 receptor modulating [3H]ACh release from rat atria

We have reported previously that norepinephrine (NE) and epinephrine reduce acetylcholine (ACh) overflow from superfused rat atria apparently through interaction with a presynaptic alpha-1 receptor. To characterize further this novel alpha-1-mediated effect, we tested the ability of a series of alpha antagonists and agonists to modulate ACh release in this preparation. The alpha-1 selective antagonists YM 12617 and WB 4101 blocked the inhibitory action of NE with IC50 values of about 0.1 and 1 nM, respectively, whereas the alpha-2 selective antagonists Wy 26703 and rauwolscine were much less potent. These data are consistent with the involvement of an alpha-1 receptor in the response to NE. ACh release was diminished by (-)-alpha-methyl-NE but similar concentrations of the alpha-2 selective agonists B-HT 920 and UK 14304 had no effect on ACh release. A number of alpha-1 selective agonists including amidephrine, cirazoline, St 587 and SK&F 89748 failed to inhibit [3H]ACh release or had only a small effect (phenylephrine). When tested as antagonists, however, phenylephrine, cirazoline and SK&F 89748 could block the inhibitory effect of NE at concentrations consistent with their affinities at alpha-1 receptors in other systems. These compounds thus bind to but do not activate the alpha receptor regulating ACh release, apparently due to their low efficacies compared to NE. Experiments carried out after alpha receptor inactivation with phenoxybenzamine demonstrate that there is little receptor reserve for the inhibition of ACh release by NE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spermidine reverses arcaine's inhibition of N-methyl-D-aspartate-induced hippocampal [3H]norepinephrine release

The inhibition of N-methyl-D-aspartate (NMDA)-induced [3H]norepinephrine ([3HNE) release by a putrescine analog was studied. We report that arcaine, diguanidinobutane, a putative competitive polyamine antagonist, completely and noncompetitively antagonized NMDA-induced [3H]NE release from rat hippocampal minces with an IC50 value of 102 microM. Arcaine did not alter kainate- or potassium-induced [3H]NE release suggesting a specific effect on NMDA-mediated responses. Spermidine did not alter NMDA-induced [3H]NE release, nor did it reverse the effect of arcaine when introduced in a normal physiologic superfusion buffer. However, spermidine reversed the effect of arcaine when superfusing with buffer that contained 5% (v/v) of the organic solvent dimethylsulfoxide. This finding suggests that the polyamine site may be located at the intracellular surface of the cell membrane. Our results provide the first evidence for polyamine modulation of the NMDA receptor ionophore complex in a functional physiologic system.     

Pharmacological and autoradiographic discrimination of sigma and phencyclidine receptor binding sites in brain with (+)-[3H]SKF 10,047, (+)-[3H]-3-[3-hydroxyphenyl]-N-(1-propyl)piperidine and [3H]-1-[1-(2-thienyl)cyclohexyl]piperidine

The benzomorphan opioid, SKF 10,047, is the prototypical agonist for the sigma receptor. In this study, pharmacological and autoradiographic analyses reveal that (+)-[3H]SKF 10,047 labels two sites in brain: a high affinity site resembling the sigma receptor and a second site, labeled with lower affinity by (+)-[3H] SKF 10,047, similar to the phencyclidine (PCP) receptor. The drug specificity of the high affinity site for (+)-[3H]SKF 10,047 resembles that of the putative sigma receptor labeled with (+)-[3H]-3-[3-hydroxyphenyl]-N-(1-propyl)piperidine [(+)-[3H]-3-PPP], being potently inhibited by (+)-3-PPP, haloperidol and (+/-)-pentazocine, and demonstrating stereoselectivity for the (+)-isomer of SKF 10,047. In contrast, these drugs are weak in inhibiting binding of (+)-[3H]SKF 10,047 to the low affinity site, whereas PCP analogs, such as 1-[1-(2-thienyl)cyclohexyl]piperidine (TCP) and 1-[1-(m-aminophenyl)cyclohexyl]piperidine (m-NH2-PCP), are potent inhibitors. No stereoselectivity for the isomers of SKF 10,047 is noted at the low affinity binding site. Autoradiographic localizations of high affinity (+)-[3H]SKF 10,047 binding sites closely resemble those of (+)-[3H]-3-PPP labeled sites with high levels of binding in the hippocampal pyramidal cell layer, hypothalamus, pontine and cranial nerve nuclei and cerebellum. By contrast, low affinity (+)-[3H]SKF 10,047 sites are most abundant in nonpyramidal layers of the hippocampus, the cerebral cortex and thalamic nuclei, similar to the distribution of [3H]TCP labeled PCP receptors.     

Characterization of a tropane radioligand, [(3)H]2beta-propanoyl-3beta-(4-tolyl) tropane ([(3)H]PTT), for dopamine transport sites in rat brain

PTT (2beta-propanoyl-3beta-[4-tolyl] tropane) is a tropane analog relatively selective for dopamine transporters in binding and uptake assays in vitro, with long-acting psychostimulant properties in vivo. To explore its utility in binding to dopamine transporters, [(3)H]PTT was synthesized and assayed for binding in rat striatal membranes and by in vitro autoradiography. In membranes, binding of [(3)H]PTT was saturable to a single class of binding sites with a K(D) value of 3 nM. The pharmacology of [(3)H]PTT binding in striatal membranes was consistent with that of a ligand selective for dopamine transporters, with dopamine-selective compounds being significantly more potent in displacing [(3)H]PTT binding than those for 5-HT or norepinephrine transporters. Although the ability of various transporter inhibitors to displace both [(125)I]RTI-55 and [(3)H]PTT binding correlated significantly with each other, there was a better correlation of inhibitor potencies versus [(3)H]PTT binding and dopamine uptake than versus [(125)I]RTI-55 binding and dopamine uptake. The differences in correlations were most noticeable for compounds relatively selective at the 5-hydroxytryptamine (serotonin) transporter. The autoradiographic distribution of [(3)H]PTT binding in coronal sections was consistent with the known distribution of the dopamine transporter, with high levels of binding evident in caudate nucleus, nucleus accumbens, and olfactory tubercle. Moderate densities of [(3)H]PTT binding were also observed in substantia nigra pars compacta, and ventral tegmental area, as well as in the anterior cingulate cortex and portions of the hypothalamus. In addition, nonspecific binding was less than 5% of total binding. Thus, [(3)H]PTT provides an accurate and convenient marker for the dopamine transporter.     

Modulation of amphetamine-stimulated [3H]dopamine release from rat pheochromocytoma (PC12) cells by sigma type 2 receptors

An important regulatory mechanism of synaptic dopamine (DA) levels is activation of the dopamine transporter (DAT), which is a target for many drugs of abuse, including amphetamine (AMPH). sigma receptors are located in dopaminergic brain areas critical to reinforcement. We found previously that agonists at sigma2 receptors enhanced the AMPH-stimulated release of [3H]DA from slices of rat caudate-putamen. In the present study, we modeled this response in undifferentiated pheochromocytoma-12 (PC12) cells, which contain both the DAT and sigma2 receptors but not neural networks that can complicate investigation of individual neuronal mechanisms. We found that enhancement of AMPH-stimulated [3H]DA release by the sigma agonist (+)-pentazocine was blocked by sigma2 receptor antagonists. Additionally, the reduction in the effect of (+)-pentazocine by the inclusion of ethylene glycol bis(beta-aminoethyl ether)-N,N,N', N'-tetraacetic acid led us to hypothesize that sigma2 receptor activation initiated a Ca2+-dependent process that resulted in enhancing the outward flow of DA via the DAT. The source of Ca2+ required for the enhancement of reverse transport did not appear to be via N- or L-type voltage-dependent Ca2+ channels, because it was not affected by nitrendipine or omega-conotoxin. However, two inhibitors of Ca2+/calmodulin-dependent protein kinase II blocked enhancement in AMPH-stimulated release by (+)-pentazocine. Our findings suggest that sigma2 receptors are coupled to the DAT via a Ca2+/calmodulin-dependent protein kinase II transduction system in PC12 cells, and that sigma2 receptor antagonists might be useful in the treatment of drug abuse by blocking elevation of DA levels via reversal of the DAT.     

Inhibitory effects of amphetamine on potassium-stimulated release of [3H]dopamine from striatal slices and synaptosomes

Amphetamine, 10(-7) M or greater, evoked the release of [3H]dopamine ([3H]DA) and inhibited subsequent K+-evoked [3H]DA release from striatal synaptosomes superfused at a flow rate (1 ml/min) that prevented reuptake. Amphetamine inhibited the K+-evoked release of [3H]DA to a lesser extent in striatal slices or in synaptosomes superfused at a flow rate (0.35 ml/min) that allowed reuptake. The observed decrease in amphetamine inhibition of K+-evoked release was primarily due to amphetamine blocking [3H]DA reuptake. Interneuronal interactions may account for some of the inhibitory effects of amphetamine on K+-evoked release in the slice. Inhibition of K+-evoked release from either slices or synaptosomes was still evident when 10(-6) M amphetamine was removed from the superfusion buffer and the spontaneous release had returned to control levels. The presence of Ca++ during amphetamine exposure was required for subsequent inhibition of K+-evoked release in synaptosomes. Amphetamine in the presence of Ca++ did not affect the subsequent release of [3H]DA evoked by the Ca++ ionophore, A23187. Therefore, amphetamine inhibition of the K+-evoked release of [3H]DA cannot be explained by prior depletion of Ca++-releasable pools. Nifedipine, 1 microM, failed to block either the Ca++-dependent release of [3H]DA or the inhibition of K+-evoked release by amphetamine. However, 1 mM cobalt inhibited the Ca++-dependent release of [3H]DA by amphetamine and antagonized the inhibition of K+-evoked release after amphetamine exposure. This suggests that amphetamine may open voltage-dependent Ca++ channels sensitive to cobalt but not nifedipine. Amphetamine may desensitize these voltage-dependent Ca++ channels and inhibit their activation by K+ depolarization.