Opioid receptor-mediated inhibition of evoked catecholamine release from cultured neurons of rat ventral mesencephalon and locus coeruleus

The effects of selective opioid agonists on the evoked release of [³H]dopamine and [³H]noradrenaline were studied in cultured dopaminergic neurons of the ventral mesencephalon (containing the substantia nigra and ventral tegmental area) and in cultured neurons of the noradrenergic locus coeruleus, respectively. The cultures were prepared from embroyonic day 15 rat brains. After 9 days in culture, the calcium-dependent release of [³H]dopamine from dopaminergic substantia nigra/ventral tegmental aera neurons induced by 23 mM k⁺ appeared to be inhibited exclusively by activation of κ-opioid receptors, as [³H]dopamine release was inhibited selectively by the κ- agonists U69,593 and dynorphin-(1–13) (EC₅₀ 8 and 5 nM, respectively), and this inhibitory effect was antagonized by the κ-selective antagonist nor-binaltorphine (K_i 0.07 nM). In contrast, cultured noradrenergic locus coeruleus neurons appeared to contain release-inhibitory μ-opioid receptors only, as evoked [³H]noradrenaline release was inhibited selectively by the μ agonist [D-Ala², MePhe⁴, Gly-ol⁵]enkephalin (EC₅₀ 45 nM), a response that was antagonized by the preferential μ antagonist naloxone (K_i = 0.7 nM). The δ-opioid receptor agonist [D-Ser²(O-butyl), Leu⁵]enkephaly-Thr⁶ did not affect catecholamine release. Dopamine release from cultured ventral mesencephalic neurons, induced by 100 μM N-Methyl-D-Aspartate (NMDA), also appeared to be subject to κ receptor-mediated inhibition, whereas NMDA-induced noradrenaline release from cultured locus coeruleus neurons was under the inhibitor control of μ receptors. It is therefore concluded that in rat brain neurotransmitter release from dopaminergic and noradrenergic neurons, originating from the substantia nigra/vental tegmental area and the locus coeruleus, is liable to inhibition by homogenous populations of κ- and μ-opioid receptors, respectively, independent of the input of non-opioid neurons from distict nuclei.     

The effects of receptor selective opioid peptides on morphine-induced analgesia

Utilizing the mouse tail-flick assay, four opioid peptides, which have been reported to be selective for either μ- or δ-opioid receptors, were examined for their analgesic potency and for their ability to modify morphine-induced analgesia. [D-Ala²,D-Leu⁵]enkephalin and [D-Ser²,Thr⁶]leucine-enkephalin, putative δ-receptor selective peptides, produced a potent analgesic response and at subanalgesic doses potentiated morphine-induced analgesia. Morphiceptin and [D-Ala²,Pro⁵]enkephalinamide, putative μ-receptor selective peptides, were similarly found to produce analgesia. However, in contrast to the δ-receptor selective peptides, three μ-receptor selective peptides were unable to alter the potency of morphine. Thus, it would appear that the potentiation of morphine analgesia is a unique property of δ-receptor selective peptides.     

Lobeline inhibits nicotine-evoked [H]dopamine overflow from rat striatal slices and nicotine-evoked Rb efflux from thalamic synaptosomes

The present study evaluated the interaction of lobeline with neuronal nicotinic acetylcholine receptors using two in vitro assays, [³H] overflow from [³H]dopamine ([³H]DA)-preloaded rat striatal slices and ⁸⁶Rb⁺ efflux from rat thalamic synaptosomes. To assess agonist interactions, the effect of lobeline was determined and compared to S(−)-nicotine. To assess antagonist interactions, the ability of lobeline to inhibit the effect of S(−)-nicotine was determined. Both S(−)-nicotine (0.1–1 μM) and lobeline (>1.0 μM) evoked [³H] overflow from superfused [³H]DA-preloaded striatal slices. However, lobeline-evoked [³H] overflow is mecamylamine-insensitive, indicating that this response is not mediated by nicotinic receptors. Moreover, at concentrations (<1.0 μM) which did not evoke [³H] overflow, lobeline inhibited S(−)-nicotine (0.1–10 μM)-evoked [³H] overflow, shifting the S(−)-nicotine concentration–response curve to the right. S(−)-Nicotine (30 nM–300 μM) increased (EC₅₀ VALUE=0.2 μM) ⁸⁶Rb⁺ efflux from thalamic synaptosomes. In contrast, lobeline (1 nM–10 μM) did not evoke ⁸⁶Rb⁺ efflux, and the lack of intrinsic activity indicates that lobeline is not an agonist at this nicotinic receptor subtype. Lobeline completely inhibited (IC₅₀ VALUE=0.7 μM) ⁸⁶Rb⁺ efflux evoked by 1 μM S(−)-nicotine, a concentration which maximally stimulated ⁸⁶Rb⁺ efflux. Thus, the results of these in vitro experiments demonstrate that lobeline inhibits the effects of S(−)-nicotine, and suggest that lobeline acts as a nicotinic receptor antagonist.     

Involvement of endogenous opioid peptides in the antinociception induced by the novel dermorphin tetrapeptide analog amidino-TAPA

The antinociceptive effect of i.t. administered N-amidino-Tyr-d-Arg-Phe-β-Ala (amidino-TAPA), an N-terminal tetrapeptide analog of dermorphin, was characterized in ddY mice. In the opioid receptor ligand-binding assays using mouse brain membranes, amidino-TAPA showed a very high affinity for μ-opioid receptors, a low affinity to δ-opioid receptors and no affinity for κ-opioid receptors. In the mouse tail-flick test, i.t. treatment with amidino-TAPA produced a potent antinociception. The antinociception induced by amidino-TAPA was significantly attenuated by i.t. pretreatment with the μ-opioid receptor antagonist β-funaltrexamine, the κ-opioid receptor antagonist nor-binaltorphimine and the δ-opioid receptor antagonist naltrindole. Moreover, the antinociception induced by amidino-TAPA was significantly attenuated by i.t. pretreatment with antisera against the endogenous κ-opioid peptides dynorphin A, dynorphin B and -neo-endorphin; and the endogenous δ-opioid peptide [Leu⁵]enkephalin. In mice lacking prodynorphin, the precursor of the endogenous κ-opioid peptides, the antinociceptive effect of amidino-TAPA was significantly attenuated compared to that in wild-type C57BL/6J mice. However, there was no difference in G-protein activation by amidino-TAPA in the spinal cord membranes from prodynorphin knockout mice and C57BL/6J mice. The present results suggest that the spinal antinociception induced by the μ-opioid receptor selective peptide amidino-TAPA is mediated in part by the release of endogenous opioid peptides in the spinal cord, which is caused by the direct stimulation of μ-opioid receptors.     

The putative polyamine antagonists ifenprodil and SL 82.0715 enhance dopamine efflux from rat striatal slices independent of NMDA receptor activation

NMDA stimulated the release of endogenous or tritiated dopamine from rat striatal slices and tritiated norepinephrine from cortical and hippocampal slices. The putative polyamine antagonists ifenprodil and SL 82.0715 inhibited [³H]norepinephrine release from cortical and hippocampal slices but enhanced the basal efflux of endogenous and tritiated dopamine from striatal slices. Incubation of striatal slices in a calcium-free buffer did not ameliorate these effects suggesting that the increase in dopamine efflux was not due to a calcium-dependent release process. Superfusion of striatal slices with 10 μM of either ifenprodil, cocaine, or amphetamine resulted in a significant release of tritiated dopamine which was reversed when the slices were again superfused with non-drug-containing buffer. The release observed in the presence of 10 μM ifenprodil (7-fold increase over basal) was intermediate between that observed for cocaine (3-fold increase) and amphetamine (12-fold increase). Both ifenprodil and SL 82.0715 also blocked the uptake of [³H]dopamine into striatal synaptosomes with IC₅₀ values of approximately 1.5 μM. This was again intermediate between the values obtained for cocaine (0.43 μM) and amphetamine (4.2 μM). These results suggest that ifenprodil and its analog SL 82.0715 may act as indirect dopamine agonists by both blocking presynaptic dopamine uptake and by directly increasing the basal efflux of dopamine.     

Differential Mechanisms Involved in the Effect of Nicotinic Agonists DMPP and Lobeline to Release [H]5-HT from Rat Hippocampal Slices

In the present study we investigated the effect of different nicotinic agonists (dimethylphenylpiperazinium-iodide (DMPP), (−)nicotine, cytisine, (−)-lobeline, and (−)epibatidine) and antagonists (mecamylamine and dihydro-β-erythroidine) on the release of [³H]5-HT from hippocampal slices. The nicotinic agonists DMPP and lobeline and electrical field stimulation, released [³H]5-HT from the hippocampus; other nicotinic agonists, such as (−)-nicotine, cytisine, and (−)-epibatidine had no effect. Unlike lobeline-induced release of [³H]5-HT, the effect of DMPP (10 and 40 μM) was antagonized by mecamylamine (20 and 10 μM). The effect of DMPP was [Ca²⁺]_o-independent. In experiments carried out at 7°C, i.e. the membrane carrier proteins are inhibited and the release by lobeline was abolished while the DMPP-induced release of 5-HT was rather potentiated. It is proposed that the effect of DMPP and lobeline, to enhance the release of [³H]5-HT from the hippocampus, was mediated by two different mechanisms. While DMPP-induced 5-HT release can be linked to a non-classical nAChR activation ([Ca²⁺]_o-independence), the effect of lobeline was likely mediated by uptake carriers. © 1997 Elsevier Science Ltd. All rights reserved.     

Evidence for differential modulation of μ-opioid receptor-mediated antinociceptive and antitussive activities by spleen-derived factor(s) from diabetic mice

The effects of spleen-derived factor(s) from diabetic mice on the antinociceptive and antitussive effects of μ-opioid receptor agonists were examined in mice. The antinociceptive effects were examined 1 week after adoptive transfer of the supernatant of spleen cell homogenate (SSCH) from diabetic mice (SSCH-D). Naive mice which had been injected with SSCH-D were less sensitive to the antinociceptive effects of μ-opioid receptor agonists, such as morphine and [ -Ala², N-MePhe⁴, Gly-ol⁵]enkephalin (DAMGO), than mice which had been injected with SSCH from non-diabetic mice. The antinociceptive effects of DAMGO was also significantly lower in naive mice injected with SSCH-D than in SSCH from non-diabetic mice (SSCH-ND)-treated naive mice, when assessed 2 weeks after adoptive transfer of SSCH. The sensitivity to the antinociceptive effect of [ ]-Pen^2,5]enkephalin (DPDPE), a δ-opioid receptor agonist, was significantly enhanced 2 weeks, but not 1 week, after adoptive transfer of SSCH-D. On the other hand, adoptive transfer of SSCH-D to naive mice had no significant effect on the recipients' antitussive sensitivities to morphine and DAMGO when assessed 1 week after transfer of SSCH-D. However, when the antitussive effect was assessed 2 weeks after adoptive transfer of SSCH, the antitussive effect of DAMGO was significantly lower in naive mice injected with SSCH-D than in SSCH-ND-treated naive mice. The reduction in the antitussive effect of DAMGO in naive mice had been injected with SSCH-D 2 weeks before testing was abolished when they were pretreated with naltrindole, a selective δ-opioid receptor antagonist. These findings suggest that some factor(s) derived from spleen cells may primarily contribute to the reduction in the antinociceptive effects, but not the antitussive effects, of supraspinal μ-opioid receptors. Furthermore, the dysfunction of μ-opioid receptors may up-regulate δ-opioid receptors, which would reduce μ-opioid receptor-mediated antitussive effects.     

Binding of [H](+)-BW373U86 to δ-opioid receptors in rat brain membranes

A tritiated form of the non-peptidic δ-opioid receptor agonist (+)-BW373U86 ((+)-4-((-R)--((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl)-N,N-diethylbenzamide) was synthesized and its binding characteristics studied. [³H](+)-BW373U86 bound with subnanomolar affinity to rat brain membranes and was displaced most effectively by ligands selective for δ-opioid receptors. Naltrindole, naltriben, and 7-benzylidenenaltrexone exhibited apparent inhibition constants of 0.06, 1.54, and 4.49 nM, respectively, while μ- or κ-selective ligands showed little affinity for this site. [³H](+)-BW373U86 binding was sensitive to the presence of guanine nucleotides; GDP caused a 3-fold decrease and 5′-guanylyl-imidodiphosphate (Gpp[NH]p) caused a 25% increase in binding affinity.     

Relationship between receptor affinity and topography of N-terminally extended and bridged [Tyr → Asp]deltorphin C analogues: Novel probes for the δ-opioid receptor

Receptor binding of N-terminally extended Tyr¹ deltorphin C analogues diminished δ and μ affinities, but with only a moderate loss in δ selectivity. Pseudopeptide bridged [Tyr¹ → Aps⁴]deltorphin C analogues drastically decreased δ affinity to yield peptides with poor δ selectivity. Low energy conformers of the peptides revealed that the bridge modifies the spatial orientation of the backbone of the N- and C-terminal sequences with respect to deltorphin C. The data indicate that the δ receptor site can accommodate an opioid peptide containing an N-terminal aliphatic extension on amino-Tyr¹, but not a heptapeptide conformationally constrained between residues 1 and 4.     

Centrally administered ß;-endorphin produces prolonged changes in δ-opioid ligand activity in vivo

Spontaneous reflex bladder contractions were recorded isometrically in urethane anesthetized rats. Bladder contractions were depressed by intracerebroventricular injections of the μ-opioid receptor agonist [D-A'la²,MePhe⁴,Gly(ol)⁵]enkephalin (DAGO) and the δ-agonist [2D-penicillamine,5D-penicillamine]enkephalin (DPDPE) respectively. The effect of DPDPE was selectively antagonized by ICI 174,864 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH; Aib = -aminoisobutyric acid). However following the administration of β-endorphin the antagonistic action of ICI 174,864 could no longer be observed. In addition ICI 174,864 exhibited agonistic activity following β-endorphin and the effects of DPDPE were prolonged in a dose related manner by β-endorphin. These observations suggest that β-endorphin may produce complex changes in central δ-opioid receptor activity.     

Chronic κ-opioid receptor antagonism delays the rise in blood pressure in spontaneously hypertensive rats

A 4-week subcutaneous treatment with the κ-opioid receptor antagonists, MR 2266 or MR 1452, delayed the age-dependent increase in systolic blood pressure in young spontaneously hypertensive rats (SHR), but not in normotensive rats. The content of β-endorphin, [Leu⁵]enkephalin, and catecholamines in various brain tissues was not affected by MR 2266. Specifity of the κ-opioid receptor antagonism was tested in SHR by injection of the κ-opioid agonist, MR 2033, and the μ agonist, morphiceptin, after 28 days of MR 1452 or saline and 2 days after the end of treatment. The increase in mean arterial blood pressure (MAP) after MR 2033 did not differ between the two groups at the 28th day of treatment but was higher 2 days later in the previous MR 1452 group, possibly indicating up-regulation of κ-opioid receptors. Morphiceptin did not alter MAP during or after MR 1452. It is concluded that κ-opioid receptors may have a tonic influence on the age dependent increase of blood pressure in SHR but not in normotensive rats.     

Alpha and beta nicotinic acetylcholine receptors subunits and synaptophysin in putamen from Parkinson's disease

It is well established that nicotinic receptors in the mammalian striatum are involved in modulation of the release of several neurotransmitters, including dopamine. In addition, nicotinic receptors with high affinity for agonists have generally been found to be reduced in the striatum in Parkinson's disease. In the present study antibodies have been used to examine which subunits contribute to the striatal nicotinic receptor loss in Parkinson's disease, and whether the reduction in [³H]nicotine binding correlates with synaptic loss. Autopsy tissue from the putamen of 12 Parkinson's disease cases and 12 age-matched control subjects was analysed by immunoblotting using antibodies against recombinant peptides specific for 3, 4, 7, β2 and β4 nicotinic acetylcholine receptor (nAChR) subunits and the synaptic marker synaptophysin, in conjunction with assessment of [³H]nicotine binding by autoradiography. The data indicate that there is no loss of 3, 4, 7 and β2 immunoreactivity in the putamen in Parkinson's disease, despite a highly significant reduction in [³H]nicotine binding. An intense signal of β4 immunoreactivity was found in human dorsal root ganglia, but not in temporal cortex or putamen samples. Synaptophysin immunoreactivities were also similar in Parkinson's disease and control cases. These results suggest that the loss of nicotine binding in the putamen in Parkinson's disease may involve an nAChR subunit (e.g., 5 and/or 6) other than those investigated. Alternatively, the results could reflect impaired subunit assembly at the plasma membrane.     

SB-616234-A (1-[6-(cis-3,5-dimethylpiperazin-1-yl)-2,3-dihydro-5-methoxyindol-1-yl]-1-[2'methyl-4'-(5-methyl-1,2,3-oxadiazol-3-yl)biphenyl-4-yl]methanone hydrochloride): a novel, potent and selective 5-HT1B receptor antagonist

SB-616234-A possesses high affinity for human 5-HT_1B receptors stably expressed in Chinese hamster ovary (CHO) cells (pK_i 8.3 ± 0.2), and is over 100-fold selective for a range of molecular targets except h5-HT_1D receptors (pK_i 6.6 ± 0.1). Similarly, affinity (pK_i) for rat and guinea pig striatal 5-HT_1B receptors is 9.2 ± 0.1. In [³⁵S]-GTPγS binding studies in the human recombinant cell line, SB-616234-A acted as a high affinity antagonist with a pA₂ value of 8.6 ± 0.2 whilst providing no evidence of agonist activity in this system. In [³⁵S]-GTPγS binding studies in rat striatal membranes, SB-616234-A acted as a high affinity antagonist with an apparent pK_B of 8.4 ± 0.5, again whilst providing no evidence of agonist activity in this system. SB-616234-A (1 μM) potentiated electrically stimulated [³H]-5-HT release from guinea pig and rat cortical slices (S₂/S₁ ratios of 1.8 and 1.6, respectively). SB-616234-A (0.3–30 mg kg⁻¹ p.o.) caused a dose-dependent inhibition of ex vivo [³H]-GR125743 binding to rat striatal 5-HT_1B receptors with an ED₅₀ of 2.83 ± 0.39 mg kg⁻¹ p.o. Taken together these data suggest that SB-616234-A is a potent and selective 5-HT_1B autoreceptor antagonist that occupies central 5-HT_1B receptors in vivo following oral administration.     

Opioid receptor types and antinociceptive activity in chronic inflammation: bothe κ- and μ-opiate agonistic effects are enhanced in arthritic rats

The antinociceptive effects obtained in arthritic rats with morphine, the opioid μ-agonist DAGO 9D-Al²,MePhe⁴,Gly-ol⁵ol⁵]enkephalin, the δ-selective agonist DTLET [D-Thr²,Leu⁵]enkephalyl-Thr, and the κ-agonist U-50,488H were compared to their corresponding effects in normal animals and morphine-pretreated arthritic rats, respectively, using a paw pressure test. The effects of the μ- and κ-agonists were increased in arthritic rats. While morphine-treated rats were cross-tolerant to the μ- and κ-agonists, no tolerance to the δ-selective agonist was found. The possibility that the potent action of morphine on this model for chronic inflammatory pain is mediated partly through κ-mechanisms is discussed.     

Binding of [3H]MSX-2 (3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine) to rat striatal membranes--a new, selective antagonist radioligand for A(2A) adenosine receptors.

The present study describes the preparation and binding properties of a new, potent, and selective A_2A adenosine receptor (AR) antagonist radioligand, [³H]3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine ([³H]MSX-2). [³H]MSX-2 binding to rat striatal membranes was saturable and reversible. Saturation experiments showed that [³H]MSX-2 labeled a single class of binding sites with high affinity (K_d=8.0 nM) and limited capacity (B_max=1.16 fmol·mg⁻¹ of protein). The presence of 100 μM GTP, or 10 mM magnesium chloride, respectively, had no effect on [³H]MSX-2 binding. AR agonists competed with the binding of 1 nM [³H]MSX-2 with the following order of potency: 5′-N-ethylcarboxamidoadenosine (NECA)>2-[4-(carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine (CGS-21680)>2-chloroadenosine (2-CADO)>N⁶-cyclopentyladenosine (CPA). AR antagonists showed the following order of potency: 8-(m-bromostyryl)-3,7-dimethyl-1-propargylxanthine (BS-DMPX)>1,3-dipropyl-8-cyclopentylxanthine (DPCPX)>(R)-5,6-dimethyl-7-(1-phenylethyl)-2-(4-pyridyl)-7H-pyrrolo[2,3-d]pyrimidine-4-amine (SH-128)>3,7-dimethyl-1-propargylxanthine (DMPX)>caffeine. The K_i values for antagonists were in accordance with data from binding studies with the agonist radioligand [³H]CGS21680, while agonist affinities were 3–7-fold lower. [³H]MSX-2 is a highly selective A_2A AR antagonist radioligand exhibiting a selectivity of at least two orders of magnitude versus all other AR subtypes. The new radioligand shows high specific radioactivity (85 Ci/mmol, 3150 GBq/mmol) and acceptable nonspecific binding at rat striatal membranes of 20–30%, at 1 nM.     

The neuropeptide FF analogue, 1DMe, acts as a functional opioid autoreceptor antagonist in the rat spinal cord

We assessed the possible influence of a neuropeptide FF analogue, 1DMe ([ -Tyr¹,(NMe)Phe³]neuropeptide FF), on the inhibitory action of endogenous and exogenous ∂-opioid receptor agonists on K⁺-evoked [Met⁵]-enkephalin release from superfused rat spinal cord slices. 1DMe (0.1–10 μM) dose-dependently enhanced the increase in superfusate [Met⁵]-enkephalin content due to the peptidase inhibitors thiorphan (1 μM) and bestatin (20 μM), and prevented the reduction in [Met⁵]-enkephalin release due to stimulation of ∂ receptors by 1 μM deltorphin I. Because it had the same effects as ∂-opioid receptor antagonists, 1DMe might act through the functional blockade of presynaptically located ∂-opioid autoreceptors.     

Acetylcholine receptor agonists stimulate [H]taurine release from rat sympathetic ganglia

The endogenous taurine content, and the uptake and release of [³H]taurine were examined using the rat superior cervical ganglion. Taurine was found to be one of the most abundant amino acids in the superior cervical ganglion, and the superior cervical ganglion took up [³H]taurine from the incubation medium. Carbachol stimulated the release of [³H]taurine in a concentration-dependent manner with an EC₅₀ of 26 μM and maximal stimulation at 100 μM. The nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium stimulated release with the same potency but with greater efficacy than carbachol. The nicotinic receptor antagonist hexamethonium (1 mM) inhibited carbachol-stimulated release by 74% (±)-Muscarine stimulated release with an EC₅₀ of 8 μM but with a maximal effect of only 32% of that produced by 100 μM carbachol. Oxotremorine another muscarinic receptor agonist, was ineffective, even at 1 mM. The muscarinic receptor antagonist atropine inhibited carbachol-stimulated release by 30% at 10μM. These results show that ³H taurine release from rat superior cervical ganglion can be stimulated by cholinergic receptor agonists. Release is mediated predominantly by a nicotinic receptor and partially by a muscarinic receptor.     

Nicotinic-agonist stimulated (86)Rb(+) efflux and [(3)H]epibatidine binding of mice differing in beta2 genotype

Nicotinic acetylcholine receptor function and binding was measured in 12 brain regions from mice differing in β2 subunit expression. Function was measured by on-line detection of ⁸⁶Rb⁺ efflux stimulated under conditions that measure two pharmacologically distinct nicotinic responses: (1) stimulation with 10 μM nicotine, a response that is relatively sensitive to inhibition by the antagonist, dihydro-β-erythroidine (DHβE); and (2) stimulation with 10 μM epibatidine in the presence of 2 μM DHβE, a response that is relatively resistant to inhibition by DHβE. Deletion of the β2 subunit profoundly reduced both DHβE-sensitive and -resistant ⁸⁶Rb⁺ efflux in each brain region and essentially eliminated activity in regions such as cerebral cortex and thalamus. However, residual activity was observed in regions such as olfactory bulbs and inferior colliculus. [³H]Epibatidine binding was measured under conditions that allow estimation of both high- and low-affinity sites. High-affinity sites sensitive to inhibition by the nicotinic agonist, cytisine, were virtually eliminated in every region by the β2 null mutation. In contrast, only a subset of the high-affinity sites insensitive to inhibition by cytisine were eliminated in β2 null mutants, suggesting receptor heterogeniety. Similarly, low affinity [³H]epibatidine binding was heterogeneous in that a fraction of the sites required the β2 subunit. Many remaining sites were sensitive to inhibition by -bungarotoxin indicating that a subset of the low affinity [³H]epibatidine binding are of the 7* subtype. Distinct regional variation was observed among the 12 brain regions. These studies confirm important roles for β2-containing receptors in mediating pharmacologically distinct functions and as components of several identifiable binding sites.     

Selective labelling of 5-HT7 receptor recognition sites in rat brain using [H]5-carboxamidotryptamine

The aim of the present study was to establish a radioligand binding assay to selectively label the native 5-HT₇ receptor expressed in rat brain. In rat whole brain (minus cerebellum and striatum) homogenate, (±)-pindolol (10 μM)-insensitive [³H]5-CT ([³H]5-carboxamidotryptamine; 0.5 nM) specific binding (defined by 5-HT, 10 μM) displayed a pharmacological profile similar to the recombinant 5-HT₇ receptor, although the Hill coefficients for competition curves generated by methiothepin, ritanserin, sumatriptan, clozapine and pimozide were significantly less than unity. In homogenates of rat hypothalamus, (±)-pindolol (10 μM)-insensitive [³H]5-CT recognition sites also resembled, pharmacologically, the 5-HT₇ receptor, although pimozide still generated Hill coefficients significantly less than unity. Subsequent studies were performed in the additional presence of WAY100635 (100 nM) to prevent [³H]5-CT binding to residual, possibly, 5-HT_1A sites. Competition for this [³H]5-CT binding indicated the labelling in whole rat brain homogenate of a homogenous population of sites with the pharmacological profile of the 5-HT₇ receptor. Saturation studies also indicated that (±)-pindolol (10 μM)/WAY 100635 (100 nM)-insensitive [³H]5-CT binding to homogenates of whole rat brain was saturable and to an apparently homogenous population of sites which were labelled with nanomolar affinity (B_max=33.2±0.7 fmol mg⁻¹ protein, pK_d=8.78±0.05, mean±S.E.M., n=3). The development of this 5-HT₇ receptor binding assay will aid investigation of the rat native 5-HT₇ receptor.     

14β-Chlorocinnamoylamino derivatives of metopon: long-term μ-opioid receptor antagonists

The affinity, selectivity and antinociceptive properties of 5β-methyl-14β-(p-chlorocinnamoylamino)-7,8-dihydromorphinone (MET-Cl-CAMO) and N-cyclopropyl-methyl-5β-methyl-14β-(p-chlorocinnamoylamino)-7,8-dihydronormorphinone (N-CPM-MET-Cl-CAMO) for the multiple opioid receptors were characterized. In competition binding assays using bovine striatal membranes, both compounds inhibited the binding of 0.25 nM [³H][

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-Ala²,(Me)-Phe⁴,Gly(ol)⁵]enkephalin (DAMGO) with IC₅₀ values of less than 2 nM. Preincubation of membranes with MET-Cl-CAMO and N-CPM-MET-Cl-CAMO produced a concentration-dependent, wash-resistant inhibition of μ-opioid receptor binding. Saturation binding experiments with N-CPM-MET-Cl-CAMO showed a reduction in the number of μ-opioid binding sites without a change in affinity. In the mouse 55°C warm-water tail-flick assay, neither MET-Cl-CAMO nor N-CPM-MET-Cl-CAMO at doses up to 100 nmol produced antinociception after intracerebroventricular administration, but morphine-induced antinociception was antagonized in a time- and dose-dependent manner by both compounds. The antagonism produced by 1 nmol of either MET-Cl-CAMO or N-CPM-MET-Cl-CAMO reached a maximal effect after 24 h, and lasted up to 48 h. Analgesia mediated by δ- or κ-opioids was not altered by either compound. In summary, the data suggest that MET-Cl-CAMO and N-CPM-MET-Cl-CAMO are long-term, μ-opioid receptor antagonists, devoid of agonist properties in the mouse tail-flick assay, and that N-CPM-MET-Cl-CAMO may produce its antagonistic effects by binding irreversibly to the μ-opioid receptor.