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.     

Drug specificity and intestinal membrane localization of human organic cation transporters (OCT)

This study was performed to investigate which human organic cation transporter, hOCT1, hOCT2 or hOCT3, participates with regard to cation specificity and membrane localization in the intestinal absorption of orally available cationic drugs. Inhibition of N-[methyl-³H]4-phenylpyridinium ([³H]MPP⁺) uptake by various compounds into Caco-2 cells and into cells (HEK-293 or CHO) that were stably transfected with hOCT1, hOCT2 or hOCT3 was compared. The uptake of [³H]MPP⁺ into Caco-2 cells was inhibited by atropine, butylscopolamine, clonidine, diphenhydramine, etilefrine, quinine and ranitidine with IC₅₀ values between 6 μM and 4 mM. Transepithelial, apical to basal flux of [³H]MPP⁺ across Caco-2 cell monolayers was also strongly inhibited by these compounds. The inhibitory potency of the cationic drugs and prototypical organic cations at Caco-2 cells correlated well with the inhibitory potency measured at CHO-hOCT3 cells but much less with that at HEK-hOCT1 and -hOCT2 cells. This is functional evidence for the predominant role of hOCT3. Etilefrine and atropine were specifically transported into CHO cells by hOCT3. In Caco-2 cells, the mRNA of all three hOCT and the proteins hOCT2 and hOCT3 were detected. More importantly, immunocytochemical analyses of human jejunum revealed for the first time that hOCT3 is localized to the brush border membrane whereas hOCT1 immunolabeling was mainly observed at the lateral membranes of the enterocytes.     

Effects of L- and N-type Ca channel antagonists on excitatory amino acid-evoked dopamine release

In thc present study we tested the effect of dihydropyridine (DHP) Ca²⁺ channel antagonists and of ω-conotoxin GVIA on [³H]dopamine (DA) release evoked by the activation of excitatory amino acid (EAA) receptors in cultures of fetal rat ventral mesencephalon, in order to investigate the role of voltage-sensitive L- and N-type Ca²⁺ channels in these EAA-mediated processes. Micromolar concentrations (10–30 μM) of DHP L-type Ca²⁺ channel antagonists inhibited [³H]DA release evoked by N-methyl-D-aspartate (NMDA), kainate, quisqualate or veratridine. [³H]DA release evoked by the L-type Ca²⁺ channel agonist, Bay K 8644, was inhibited by lower concentrations (0.1–1 μM) of the DHP antagonist, nitrendipine, than was the release evoked by EAAs. The DHP antagonist, ( + )-PN 200-110, was more potent than ( − )-PN 200-110 in inhibiting [³H]DA release evoked by Bay K 8644, but the two stereoisomers were equipotent in inhibiting NMDA-evoked release. These results indicate that activation of L-type Ca²⁺ channels is able to evoke [³H]DA release. However activation of L-type channels is not involved in EAA-induced [³H]DA release and therefore inhibition of EAA-induccd [³H]DA release by micromolar concentrations of DHPs must be mediated by actions other than inhibition of L-type Ca²⁺ channels. ω-Conotoxin GVIA (3 μM) had no effect on [³H]DA release evoked by Bay K 8644, indicating that the toxin may selectively inhibit N-type channels in this preparation. ω-Conotoxin GVIA (3 μM) partially inhibited [³H]DA release evoked by NMDA or kainate, suggesting that N-type Ca²⁺ channels could possibly play a role in FAA-mediated responses in these cells.     

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.     

Nitric oxide induces neurotransmitter release from hippocampal slices

Hydroxylamine (1–300 μM), a nitric oxide generator, stimulated the release of [³H]norepinephrine ([³H]NE) and [¹⁴C]acetylchoIine ([¹⁴C]ACh) from rat hippocampal slices in a concentration-dependent manner (EC₅₀ = 30 μM). A maximally effective concentration of hydroxylamine (300 μM) produced a 24-fold increase in the basal [³H]NE and 3.6-fold increase in the in the basal [¹⁴C]ACh efflux. Sodium nitroprusside (SNP), also stimulated the release of [³H]NE, but only at high concentrations (10–30 mM). Calcium-free experimental buffer (1 mM EGTA) abolished the response. Hemoglobin (0.3 μM) inhibited the effect of 100 μM hydroxylamine in a manner which was specific for nitric oxide. In addition, 100 μM hydroxyiarnine increased the efflux of endogenous GABA and glutamate by 3- and 6-fold, respectively.     

The action of trelibet, a new antidepressive agent on [H]noradrenaline release from rabbit pulmonary artery

Trelibet, a new antidepressant, used at 10⁻⁷–10⁻⁴ M failed to affect the [³H]noradrenaline ([³H]NA) release evoked from the isolated main pulmonary artery of the rabbit low frequency (2 Hz) nerve stimulation whether the neuronal uptake inhibitor cocaine (3 × 10⁻⁵ M) was present or not. Its metabolite (EGYT-2760) however, potentiated the nerve-evoked release of [³H]NA. In the absence of cocaine both the resting and the stimulation-evoked release of ³H increased in response to EGYT-2760. These effect were accompanied by muscle contraction. The EGYT-2760-potentiated transmitter release was inhibited either by exogenously applied 1-noradrenaline (10⁻⁶ M) or clonidine (10⁻⁶ M), preferential agonists of presynaptic ₂-adrenoceptors. The 1-noradrenaline-induced inhibition of transmitter release potentiated by EGYT-2760 was antagonized by 3 × 10⁻⁷ M yohimbine, a preferential ₂-adrenoceptor inhibitor. In the absence of cocaine, Ca²⁺ removal from the external medium failed to affect the ³H outflow-increasing effect of EGYT-2760 but abolished the nerve-evoked release-potentiating action of this compound. It is concluded that the metabolite of trelibet exerts a ‘yohimbine-like’ action, as well as a ‘tyramine-like’ effect in peripheral sympathetic nerve fibres.     

A rapid screening system to determine drug affinities for the intestinal dipeptide transporter 1: system characterisation

Purpose: To establish an in vitro system for the rapid assessment of the affinities of potential substrates for the di/tri/oligopeptide transport system (DTS). Methods: Monolayers of Caco-2 cells were cultured in plastic wells for 7–9 days and the uptake of Gly-[³H]L-Pro, a specific and relatively stable substrate for the DTS was used as an affinity probe. Gly-[³H]L-Pro (50 nM), together with excess L-Pro (10 mM), to suppress uptake of any [³H]L-Pro produced by degradation of the probe, was incubated with the test compound (usually 1 mM) at pH 6 for 3 min. The uptake of radiolabel was determined by liquid scintillation counting. Results: High specific-uptake (>85%) of Gly-[³H]L-Pro was obtained with cells grown for 7–9 days. Gly-[³H]L-Pro uptake had a substantial active concentration-dependent component (K_m of 0.39±0.02 mM, V_max of 0.98±0.04 nmol min⁻¹ (mg protein)⁻¹. This process was shown to be specific for the DTS as evidenced by the significant inhibition by compounds reported to be transported by this system and the lack of inhibition by amino acids. The use of low competitor concentrations (1 mM) enabled a range of inhibition values (0–89%) of a series of competitors (amino acids, dipeptides and β-lactam antibiotics) to be estimated, illustrating that structurally similar compounds can be ranked for affinity to the DTS. Conclusion: A screening system, using Caco-2 cells and the dipeptide Gly-[³H]L-Pro as a displaceable probe, was developed to assess a variety of compounds for recognition by the di/tri/oligopeptide transport system. This fully describes the first system that allows structurally related compounds to be ranked on the basis of their affinity for the DTS recognition site.     

The effect of trimethyltin on acetylcholine release in the guinea-pig trachea

The purpose of the present work was to characterise the effects of trimethyltin on the release of acetylcholine from parasympathetic nerves and its effect on the postjunctional cholinergic stimulation of a smooth muscle. The guinea-pig trachea has been used as a model. Prejunctionally, trimethyltin (3.0 × 10⁻³ M) significantly enhanced in a reversible manner the high K⁺ (75 mM) evoked release of endogenous acetylcholine and [³H]acetylcholine. The evoked release of endogenous acetylcholine and [³H]acetylcholine was released from a pool of acetylcholine being independent of extraneuronal Ca²⁺ in the presence, but not in the absence of trimethyltin. The effect of trimethyltin on the release was not inhibited by low Ca²⁺ (0 mM and 1.0 × 10⁻⁴ M) or by Ca²⁺ channel blockers (verapamil, 1.0 × 10⁻⁴ M, flunarizine, 1.0 × 10⁻⁴ M, ω-conotoxin GVIA, 2.0 × 10⁻⁷ M and ω-agatoxin, 2.0 × 10⁻⁷ M). The present results also demonstrate that trimethyltin induce emptying of a non-vesicular, probably a cytoplasmic storage pool of acetylcholine, since AH5183 (2.0 × 10⁻⁵ M), an inhibitor of the translocation of acetylcholine into synaptic vesicles, and -latrotoxin (1.0 × 10⁻⁸ M), a toxin from black widow spider venom inducing vesicle depletion, had no inhibitory effects on the release of [³H]acetylcholine evoked by trimethyltin (3.0 × 10⁻³ M). The release of [³H]acetylcholine was moreover enhanced by trimethyltin when the vesicular uptake of [³H]acetylcholine was inhibited by AH5183, probably as a result of a higher cytoplasmic concentration of [³H]acetylcholine. Trimethyltin also reduced the neuronal uptake of [³H]choline and this was probably due to a depolarising effect of trimethyltin on the cholinergic nerve terminals. A similar depolarisation induced by trimethyltin was observed during patch clamping of GH₄ C₁ neuronal cells. Postjunctionally, trimethyltin had no effect by itself or on the carbachol-induced smooth muscle contraction, indicating that trimethyltin did not have a general depolarising effect on smooth muscle cells or an effect on muscarinic receptors. Furthermore, the reduced electrical field-induced contraction and the subsequent increase in the basal smooth muscle tension that was observed by addition of trimethyltin was activity-dependent, and was most probably due to emptying of a nervous non-vesicular storage pool of acetylcholine, followed by rapid hydrolysis of acetylcholine by acetyl- and pseudocholinesterases.     

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.     

Biochemical evidence for the 5-HT agonist properties of PAT (8-hydroxy-2-(di-n-propylamino)tetralin) in the rat brain

In vitro investigations revealed that PAT (8-hydroxy-2-(n-dipropylamino)tetralin) interacted with postsynaptic 5-HT receptors in the rat brain: the drug stimulated 5-HT-sensitive adenylate cyclase in homogenates of colliculi from new-born rats (K_Aapp 8.6 μM) and inhibited the specific binding of [³H]5-HT to 5-HT₁ sites. The PAT-induced inhibition of [³H]5-HT binding showed marked regional differences compatible with a preferential interaction of PAT (IC₅₀ 2 nM) with the 5-HT_1A subclass. As previously seen with 5-HT agonists, the efficacy of PAT for displacing [³H]5-HT bound to hippocampal membranes was markedly increased by Mn²⁺ (1 nM) and reduced by GTP (0.1 nM). PAT also affected presynaptic 5-HT metabolism since it inhibited competitively (K_i 1.4 μM) [³H]5-HT uptake into cortical synaptosomes and reduced (in the presence of the 5-HT uptake inhibitor fluoxetine) the K⁺-evoked release of [³H]5-HT previously taken up or newly synthesized from [³H]tryptophan in cortical or striatal slices. This latter effect was prevented by 5-HT antagonists (methiothepin, metergoline) suggesting that it was mediated by the stimulation of presynaptic 5-HT autoreceptors by PAT. Like 5-HT, PAT counteracted the stimulatory effect of K⁺-induced depolarization on the synthesis of [³H]5-HT from [³H]tryptophan in cortical slices. It is concluded that PAT is a potent 5-HT agonist acting on both post- and presynaptic 5-HT receptors in the rat brain.     

Selective effects of [D-Ser(O-t-butyl),Leu]enkephalyl-Thr and [D-Ser(O-t-butyl),Leu]enkephalyl-Thr(O-t-butyl), two new enkephalin analogues, on neurotransmitter release and adenylate cyclase in rat brain slices

The selectivity and potency of two new enkephalin-derived δ-opioid receptor agonists, DSTBULET ([D-Ser²(O-t-butyl), Leu⁵]enkephalyl-Thr⁶) and BUBU ([D-Ser²(O-t-butyl),Leu⁵]enkephalyl-Thr⁶(O-t-butyl)) were determined with functional tests in vitro of μ-, δ-, and κ-opioid receptor activation in the rat brain. Both peptides concentration dependently (1 nM-1 μM) inhibited the release of radiolabeled acetylcholine (ACh) from striatal slices (pD₂ 7.6–7.9), an effect exclusively mediated by δ-opioid receptor activation. Fentanyl isothiocyanate (FIT), an irreversible δ-antagonist, completely blocked the inhibitory effects of DSTBULET and BUBU. Up to a concentration of 1 μM, the peptides did not affect striatal [³H]dopamine (DA) release nor cortical [³H]noradrenaline (NA) release, processes which are known to be inhibited by opioids activating κ and μ-receptors, respectively. Furthermore, both DSTBULET and BUBU caused a strong inhibition (pD₂ 8.2–8.3) of D-1 dopamine receptor-stimulated cyclic AMP efflux from striatal slices, an effect known to be mediated by μ- and/or δ-opioid receptor activation. However, the peptides were without effect when D-1 and D-2 dopamine receptors were stimulated simultaneously, a situation in which only μ-agonists are able to inhibit the resulting cAMP efflux. In conclusion, DSTBULET and BUBU appear to display a high selectivity and potency toward functional δ-opioid receptors in the brain.     

Gamma-hydroxybutyric acid (GHB) and gamma-aminobutyric acidB receptor (GABABR) binding sites are distinctive from one another: molecular evidence

γ-Hydroxybutyric Acid (GHB) is thought to be a weak partial agonist at the γ-aminobutyric acid_B Receptor (GABA_BR), but the precise relationship of the GHB receptor (GHBR) to the GABA_BR remains unclear. In order to test the hypothesis that the GHBR is not identical to the GABA_BR, we conducted two groups of experiments. First, GABA_BR subtype 1 (R1) and/or subtype 2 (R2) were over expressed in HEK 293 cells and membrane binding studies on the transfected cells done using [³H]GHB and [³H] (2E)-(5-hydroxy-5,7,8,9-tetrahydro-6H-benzo[a][7]annulen-6-ylidene) ethanoic acid ([³H]NCS-382). The latter is a specific antagonist at the GHB binding site. Second, [³H]GHB and [³H]NCS-382 autoradiographic binding studies were done on the brains of mice in which the gene for GABA_BR1a was deleted. Such mice do not have a functioning GABA_BR. There was no detectable specific [³H]GHB or [³H]NCS-382 binding in HEK 293 cells transfected with GABA_BR1, R2, or R1/R2. Binding to [³H]CGP54626A, a high affinity GABA_BR antagonist, was absent in GABA_BR1a^−/− mice. There was no difference in [³H]NCS-382 binding observed in the brains of GABA_BR1a^−/−, GABA_BR1a^+/− or GABA_BR1a^+/+ mice. Specific [³H]GHB binding was observed in the brain of GABA_BR1a^−/− mice but was significantly lower than in wild type mice. These data support the hypothesis that the GHB binding site is separate and distinct from the GABA_BR.     

Receptor-mediated internalization of [3H]-neurotensin in synaptosomal preparations from rat neostriatum

Following its binding to somatodendritic receptors, the neuropeptide neurotensin (NT) internalizes via a clathrin-mediated process. In the present study, we investigated whether NT also internalizes presynaptically using synaptosomes from rat neostriatum, a region in which NT1 receptors are virtually all presynaptic. Binding of [³H]–NT to striatal synaptosomes in the presence of levocabastine to block NT2 receptors is specific, saturable, and has NT1 binding properties. A significant fraction of the bound radioactivity is resistant to hypertonic acid wash indicating that it is internalized. Internalization of [³H]–NT, like that of [¹²⁵I]–transferrin, is blocked by sucrose and low temperature, consistent with endocytosis occurring via a clathrin-dependent pathway. However, contrary to what was reported at the somatodendritic level, neither [³H]–NT nor [¹²⁵I]–transferrin internalization in synaptosomes is sensitive to the endocytosis inhibitor phenylarsine oxide. Moreover, treatment of synaptosomes with monensin, which prevents internalized receptors from recycling to the plasma membrane, reduces [³H]–NT binding and internalization, suggesting that presynaptic NT1 receptors, in contrast to somatodendritic ones, are recycled back to the plasma membrane. Taken together, these results suggest that NT internalizes in nerve terminals via an endocytic pathway that is related to, but is mechanistically distinct from that responsible for NT internalization in nerve cell bodies.     

Distribution of tritiated tetrodotoxin administered intraperitoneally to pufferfish

S. , Y. , M. , N. , K. , T. , N. and K. . Distribution of tritiated tetrodotoxin administered intraperitoneally to pufferfish. Toxicon 25, 1283 – 1289, 1987. — Tetrodotoxin was recoil-tritiated by the ³He(n,p)³H reaction and purified by gel filtration. The [³H]tetrodotoxin gave only one spot in both cellulose acetate strip electrophoresis and thin layer chromatography. The specific toxicity of tetrodotoxin did not decrease during the recoil tritiation and the [³H]tetrodotoxin showed a specific radioactivity of 25 × 10⁻⁶ Ci/mmole. In spite of the low specific radioactivity, the [³H]tetrodotoxin was able to be used to investigate the anatomical distribution of tetrodotoxin in pufferfish. When intraperitoneally injected into ‘torafugu’ puffer, [³H]tetrodotoxin accumulated in most tissues, the level being highest in the skin, followed by the liver, intestines and muscle. With time, the [³H]tetrodotoxin radioactivity level in the injected pufferfish decreased in most tissues, except for skin and gallbladder. Based on these results, the metabolism of tetrodotoxin in pufferfish is discussed.     

Dopamine receptors modulating [H]acetylcholine release in slices of the cat caudate: Effects of (−)-N-(2-chloroethyl)-norapomorphine

(−)-N-(2-Chloroethyl)-norapomorphine ((−)-NCA) inhibited in a concentration-dependent manner the electrically evoked [³H]acetylcholine release in slices of cat caudate. The inhibition by (−)-NCA was reversible and antagonized by the benzamide neuroleptic S-sulpiride. Although (−)-NCA is an irreversible antagonist at some behaviorally relevant postsynaptic dopamine receptors, its effect as an agonist on dopamine receptors modulating [³H]acetylcholine release strongly resembles its action on presynaptic dopamine autoreceptors modulating [³H]dopamine release. Our results suggest that the dopamine receptor modulating [³H]acetylcholine release may not be an appropriate in vitro model for those behaviorally relevant postsynaptic dopamine receptors which are antagonized by (−)-NCA. It is more likely that it conforms to the characteristics of presynaptic release-modulating dopamine autoreceptors. The agonistic action of (−)-NCA at presynaptic dopamine receptors, in contrast to the irreversible antagonism of some postsynaptic dopamine receptors by (−)-NCA, should be interpreted with caution. Evidence is presented which suggests that (−)-NCA breaks down in solution into (−)-N-(2-hydroxylethyl)-norapomorphine ((−)-NHA). Since (−)-NHA is an agonist at presynaptic dopamine receptors, this physicochemical breakdown product may be partly responsible for the apparent agonistic properties of (−)-NCA under our in vitro conditions.     

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.     

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.     

Vesicular site of action of lithium ion in choline-calcium stimulated adrenergic nerve endings of rat heart

Reports from this laboratory have suggested that the secretion of norepinephrine (NE) in slices of ventricle from the rat heart, incubated in a medium of choline and Ca²⁺ (Ch⁺-Ca²⁺) deprived of Na⁺, is mediated by the outward transport of NE (blocked by cocaine) from synaptic vesicles fused or attached to the axolemma. Lithium (and reserpine), block the Mg²⁺-ATP-stimulated uptake of NE by isolated synaptic vesicles (Slotkin, Seidler, Whitmore, Salvaggio, and Lau, 1978). Hence, a hypothesis to be tested was that lithium ions would increase secretion of NE stimulated by Ch⁺-Ca²⁺. By contrast, amines mobilized by lithium ions (or reserpine) in control nerves would be deaminated. Experiments showed that lithium-Krebs increased the excretion of [³H]deaminated metabolites of [³H]NE. A much smaller quantity of [³H]NE was released by a process that was independent of Ca²⁺ and weakly inhibited by cocaine. When combined with Ch⁺Ca²⁺, lithium ions, in concentrations that were known to block uptake in isolated vesicles, induced a Ca²⁺-dependent secretion of [³H]amines. A small quantity of [³H]deaminated metabolites was excreted. Both processes were strongly inhibited by both cocaine and desipramine. Both reserpine (Bogdanski, 1982) and lithium ions blocked the inhibitory effect of exogenous ATP (2 mM) on secretion induced by Ch⁺−Ca²⁺. This effect indicated a proximity of vesicles to the axolemma. The effects of lithium ions could be explained by the hypothesis if lithium prevented storage of [³H]NE in vesicles by blocking the Mg²⁺ + ATP-dependent amine pump in vesicle membranes.     

Development, validation and utility of an in vitro technique for assessment of potential clinical drug–drug interactions involving P-glycoprotein

Regulatory interest is increasing for drug transporters generally and P-glycoprotein (Pgp) in particular, primarily in the area of drug–drug interactions. To aid in both identifying and discharging the potential liabilities associated with drug–transporter interactions, the pharmaceutical industry has a growing requirement for routine and robust non-clinical assays. An assay was designed, optimised and validated to determine the in vitro inhibitory potency of new chemical entities (NCEs) towards human Pgp-mediated transport. [³H]-Digoxin was established as a suitable probe substrate by investigating its characteristics in the in vitro system (MDCKII-MDR1 cells grown in 24-multiwell inserts). The inhibitory potencies (apparent IC₅₀) of known Pgp inhibitors astemizole, GF120918, ketoconazole, itraconazole, quinidine, verapamil and quinine were determined over at least a 1000-fold concentration range. Validation was carried out using manual and automatic techniques. [³H]-Digoxin was found to be stable and have good mass balance in the system. In contrast to [A → B] transport, [³H]-digoxin [B → A] transport rates were readily measured with good reproducibility. There was no evidence of saturation of transport up to 10 μM digoxin and 30 nM digoxin was selected for routine assay use, reflecting clinical therapeutic concentrations. IC₅₀ values ranged over approximately 100-fold with excellent reproducibility. Results from manual and automated versions were in close agreement. This method is suitable for routine use to assess the in vitro inhibitory potency of NCEs on Pgp-mediated digoxin transport. Comparison of IC₅₀ values against clinical interaction profiles for the probe inhibitors indicated the in vitro assay is predictive of clinical digoxin–drug interactions mediated via Pgp.