Inhibition of P-glycoprotein-mediated transport by terpenoids contained in herbal medicines and natural products

Terpenoids form a large and structurally diverse family of natural products and are ingredients of various herbal medicines. We have investigated possible interactions between herbal medicines and conventional medicines, and recently reported that monoterpenoids contained in Zanthoxyli Fructus can be potent inhibitors of P-glycoprotein (P-gp). In the present study, the influence of 70 kinds of terpenoids present in natural products on P-gp-mediated efflux transport was investigated. LLC-GA5-COL150 cells transfected with human MDR1 cDNA encoding P-gp were used to screen the terpenoids. Large increases in the intracellular accumulation of [³H]digoxin were observed in the presence of (R)-(+)-citronellal, (S)-(−)-β-citronellol, -terpinene, terpinolene, (−)-β-pinene, abietic acid, ophiobolin A, cucurbitacin I, and glycyrrhetic acid. A study of the concentration-dependency revealed that the IC₅₀ of ophiobolin A, glycyrrhetic acid, (R)-(+)-citronellal, abietic acid, and cucurbitacin I was smaller than that of verapamil. The transcellular transport of [³H]digoxin across Caco-2 cell monolayers was then examined in the presence of (R)-(+)-citronellal, abietic acid, and glycyrrhetic acid. Significant increases in the apical-to-basolateral transport and decreases in the basolateral-to-apical transport and efflux ratio were demonstrated. These findings suggest that some natural products containing these terpenoids may inhibit P-gp-mediated transport and interact with P-gp substrates in the intestinal absorption process.     

Isothiazole dioxide derivative 6n inhibits vascular smooth muscle cell proliferation and protein farnesylation

Isothiazole dioxides have been shown to inhibit Trypanosoma brucei protein farnesyltransferase (PFTase) in isolated enzyme, but elicited only a minor effect on mammalian PFTase. In the present study we have evaluated the effect of 3-diethylamino-4-(4-methoxyphenyl)-isothiazole 1,1-dioxides with different substituents at C5, on rat PFTase and protein geranylgeranyltransferase-I (PGGTase-I) with the final aims to improve the potency against mammalian PFTase and to identify new compounds with antiproliferative properties. For these purposes, in vitro and cell culture models have been utilized. The results showed that isothiazole dioxides with C4–C5 double bond and sulfaryl substituted at the C5 position but none of the dihydro-derivatives, were able to inhibit in vitro PFTase in a concentration dependent manner (IC₅₀ ranging from 8.56 to 1015 μM). Among those, compound 6n (C5; methyl-S) displayed 500-fold higher inhibitory potency on PFTase than PGGTase-I. Compound 6n was shown to affect rat smooth muscle cell (SMC) proliferation at concentrations similar (IC₅₀ = 61.4 μM) to those required to inhibit [³H]-farnesol incorporation into cellular proteins (−44.1% at 100 μM). Finally, compound 6n interferes with rat SMC proliferation by blocking the progression of G0/G1 phase without inducing apoptosis, as assessed by [³H]-thymidine incorporation assay and flow cytometry analysis. Taken together, we described a new PFTase inhibitor containing the isothiazole dioxide moiety that affects mammalian protein farnesylation and SMC proliferation by inhibiting G0/G1 phase of the cell cycle.     

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.     

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.     

Neuroprotective effects of RPR 104632, a novel antagonist at the glycine site of the NMDA receptor, in vitro

The NMDA antagonist and neuroprotective effects of RPR 104632 (2H-1,2,4-benzothiadiazine-1-dioxide-3-carboxylic acid), a new benzothiadiazine derivative, with affinity for the glycine site of the NMDA receptor-channel complex are described. RPR 104632 antagonized the binding of [³H]5,7-dichlorokynurenic acid to the rat cerebral cortex, with a K_i of 4.9 nM. This effect was stereospecific, since the (-)-isomer was 500-fold more potent than the (+)-isomer. The potent affinity of RPR 104632 for the glycine site was confirmed by the observation that RPR 104632 inhibited [³H]N-(1-(2-thienyl)cyclohexyl]-3,4-piperidine ([³H]TCP) binding in the presence of N-methyl- -aspartate (NMDA) (IC₅₀ = 55 nM), whereas it had no effect on the competitive NMDA site or on the dissociative anaesthetic site. RPR 104632 inhibited the NMDA-evoked increase in guanosine 3′,5′-cyclic monophosphate (cGMP) levels of neonatal rat cerebellar slices (IC₅₀ = 890 nM) in a non-competitive manner and markedly reduced NMDA-induced neurotoxicity in rat hippocampal slices and in cortical primary cell cultures. These results suggest that RPR 104632 is a high-affinity specific antagonist of the glycine site coupled to the NMDA receptor channel with potent neuroprotective properties in vitro.     

Investigation of the inhibitory effect of broussochalcone A on respiratory burst in neutrophils

Broussochalcone A, a prenylated chalcone isolated from Broussonetia papyrifera (L.) VENT. (Moraceae), inhibited O₂ consumption in formylmethionyl-leucyl-phenylalanine (fMLP)- and phorbol 12-myristate 13-acetate (PMA)-stimulated rat neutrophils in a concentration-dependent manner with IC₅₀ values of 70.3±4.9 and 63.9±7.1 μM, respectively. Broussochalcone A did not affect the fMLP-induced increase of cellular inositol trisphosphate (IP₃) and [Ca²⁺]_i. However, the enzyme activity of neutrophil cytosolic protein kinase C was effectively suppressed by broussochalcone A. Broussochalcone A had no effect on either [³H]phorbol 12,13-dibutyrate ([³H]PDB) binding to neutrophil cytosolic protein kinase C or on PMA-induced membrane translocation of protein kinase C-β in neutrophils. Broussochalcone A suppressed the enzyme activity of trypsin-treated rat brain protein kinase C in a concentration-dependent manner. In PMA-activated neutrophil particulate NADPH oxidase, broussochalcone A attenuated superoxide anion radical (O₂⁻) generation with an IC₅₀ value of 61.8±5.4 μM. These results show that the inhibitory effect of broussochalcone A on respiratory burst in neutrophils is not mediated by the reduction of phospholipase C activity, but is mediated partly by the suppression of protein kinase C activity through interference with the catalytic region and by the attenuation of O₂⁻ generation from the NADPH oxidase complex.     

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.     

[H]Doxepin interactions with histamine H1-receptors and other sites in guinea pig and rat brain homogenates

[³H]Doxepin, a tricyclic antidepressant, binds to brain homogenates with two saturable components. The high affinity component, with a dissociation constant (K_D) of 0.26 nM, is associated with histamine H₁-receptors. This high affinity binding shows stereospecificity in that d-chlorpheniramine is 100 times more potent than the pharmacologically less active l-isomer. Its drug specificity and regional variation closely parallel those exhibited by [³H]mepyramine binding. The drug specificity of the low affinity component is distinct from that of histamine H₁-receptors, with no stereospecifityty for chlorpheniramine isomers. Furthermore, all the H₁-histamine antagonists tested display micromolar potency at the low-affinity doxepin sites but nanomolar potency at the high-affinity doxepin sites associated with a physiological histamine H₁-receptor. The drug specificity of the low affinity site does not correspond to that of any known neurotransmitter receptor. Tricyclic antidepressants display IC₅₀ values of 30–600 nM for the inhibition of [³H]doxepin binding to the low-affinity component with most values in the 0.1–0.3 μM affinity range.     

The effect of extracellular sodium ion concentration on the action of opiates to inhibit potassium-evoked released of [H]noradrenaline from the mouse vas deferens

Opiates depress the potassium-induced efflux of [³H]noradrenaline from the mouse vas deferens in a concentration-dependent (the IC₅₀ for normorphine was 1.5 μM), stereospecific and naloxone-reversible manner. As the concentration of sodium in the extracellular fluid was reduced, the inhibitory action of opiates was also reduced. This attenuation of opiate action is the converse of that predicted by the ‘sodium-shift’ observed in opiate binding studies in which lowering the sodium concentration potentiates opiate agonist binding. The relevance of sodium to the pharmacological actions of opiates is discussed.     

Interactions of salsolinol and β-carbolines with cerebral GABA/Benzodiazepine receptor complex: Alteration during alcohol dependence

Effects of salsolinol and 3-carboxy ester of β-carboline, which are known as condensation products of acetaldehyde formed in mammalian brain, on γ-amino-butyric acid (GABA)_A receptor/benzodiazepine receptor/chloride channel complex and its alterations during the development of alcohol dependence were studied. In synaptic membrane preparation, ethyl β-carboline-3-carboxylate (β-ECC) was found to bind to a different site from that for benzodiazepines. The benzodiazepine-stimulated [³H] muscimol binding to GABA_A receptor was suppressed by β-ECC, whereas [³H]β-ECC binding was found to be suppressed by GABA, muscimol and secobarbital. Gel column chromatography using Sephadex G-200 of the solubilized fraction from cerebral particulate fraction by sodium deoxycholate indicated that [³H]β-ECC binding site was eluted in the same fraction (molecular weight = 230,000) as the binding sites for [³H]muscimol, [³H]flunitrazepam, and [³H]t-butylbicycloorthobenzoate, known as a blocker of chloride channel. Salsolinol stimulated the specific bindings of [³H]flunitrazepam and [³H]β-ECC to cerebral synaptic membranes in the presence of chloride anion. GABA-stimulated ³⁶C1-influx into membrane vesicles was stimulated by flunitrazepam, while attenuated by β-ECC, and these effects of flunitrazepam and β-ECC were accentuated by salsolinol. In alcohol dependent mice, the stimulating effect of salsolinol on the bindings of [³H]flunitrazepam and [³H]β-ECC to cerebral synaptic membrane was eliminated. These results indicate that β-ECC binding site may reside on GABA_A receptor complex, and salsolinol may modulate the functions of benzodiazepine and β-ECC binding sites in the brain. The present results also suggest that the alteration of modulatory action of salsolinol on the cerebral [³H]flunitrazepam and [³H]β-ECC binding sites may be involved in the occurrence and/or maintenance of alcohol dependent conditions.     

The effect of tri-iodothyronine in combination with imipramine on [H]-cyclic AMP production in slices of rat cerebral cortex

Tri-iodothyronine (T₃; 15 μg) was administered to rats, alone or in combination with imipramine (20 mg/kg), for 5 days. The net synthesis of [³H]-cyclic AMP in cerebral cortex slices of animals so treated and control rats was then measured. The dose-dependent stimulation of [³H]-cyclic AMP by norepinephrine (NE) was significantly reduced in imipramine-treated rats. Tri-iodothyronine treatment had no effect on the enhanced net synthesis of [³H]-cyclic AMP produced by NE. In cortex slices of rats given both T₃ and imipramine, NE produced less stimulation of [³H]-cyclic AMP than in control rats. The magnitude of this inhibitory effect was less than that observed in animals treated with imipramine alone. In vitro addition of imipramine to the cortex slice preparation reduced the stimulation of [³H]-cyclic AMP caused by NE; treatment of rats with T₃ did not modify this inhibitory effect of imipramine in vitro. Isoproterenol produced significantly less stimulation of [³H]-cyclic AMP net synthesis than did NE; imipramine added in vitro had no effect on the stimulation produced by isoproterenol. It is concluded that the reason for the enhanced clinical effect of imipramine when given together with T₃ is not due to the hormone exaggerating the effect of the antidepressant on NE stimulated adenylate cyclase. Furthermore, for NE to produce maximal stimulation of [³H]-cyclic AMP, uptake of the catecholamine is necessary.     

Clonidine accumulation in human neuronal cells

After transport across several epithelial barriers including the blood–brain barrier, clonidine interacts with ₂-adrenergic receptors and imidazoline binding sites in the brain. We hypothesized that neuronal cells take up clonidine thereby removing the drug from the extracellular fluid compartment. Uptake of [³H]clonidine into SH-SY5Y neuroblastoma cells was linear for up to 1 min, unaffected by inside directed Na⁺ or Cl⁻ gradients but strongly inhibited by an outside pH of 6.0. The cells accumulated [³H]clonidine 50–70-fold uphill against a concentration gradient. Unlabeled clonidine, guanabenz, imipramine, diphenhydramine, maprotiline, quinine and the endogenous monoamine phenylethylamine (2 mM) strongly inhibited the [³H]clonidine uptake by 60–95%. Tetraethylammonium, choline and N-methyl-4-phenylpyridinium had no effect. The accumulation at pH 7.5 was saturable with an apparent Michaelis–Menten constant (K_t) of 0.7 mM. We conclude that SH-SY5Y cells not only bind clonidine to extracellular receptors but also take up the drug rapidly by a specific and concentrative mechanism.     

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.     

Cholecystokinin-8 increases K-evoked [H]γ-aminobutyric acid release in slices from various brain areas

[³H]γ-Aminobutyric acid (GABA) release was studied in rat brain slices in the absebce or presence of cholecystokinin-8 (CCK-8). [³H]GABA release under the conditions used was Ca²⁺-dependent and insensitive tot he presence of the glial uptake blocker β-alanine. While the basal release of [³H]GABA was not affected by CCK-8, the K⁺-stimulated release of [³H]GABA wasm significantly enhanced by 300 nM of CCK-8 in the caudate putamen, the substantia nigra, the hippocampal formation and the parietofrontal cortex. In the cerebral cortex the CCK-8 enhancement of [³H]GABA release was concentration-dependent and abolished by the CCK_B receptor antagonists PD135,158 (1.0 nM) and L-365,260 (100 nM). A significant counteraction of the CCK-8 action was also found with the CCK_A receptor antagonist L-364,718 (100 nM) but only in concentrations at which both CCK_A and CCK_B receptors are blocked. No CCK-8 effects on [³H]GABA release were observed when tetrodotoxin was superfused 5 min before the K⁺-induced [³H]GABA release. It is suggested that the enhancing actions of CCK-8 on K⁺-stimulated [³H]GABA release is mainly related to an activation of CCK_B receptors.     

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.     

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.     

The promiscuity of the dopamine transporter: implications for the kinetic analysis of [3H]serotonin uptake in rat hippocampal and striatal synaptosomes

Evidence indicates that monoaminergic neurotransmitter transporters are promiscuous, transporting substrates other than their cognate neurotransmitters. For example, serotonin is transported by the dopamine transporter (DAT) under conditions in which serotonin transporter (SERT) activity is eliminated (e.g., pharmacological inhibition). We performed a kinetic analysis of [³H]serotonin uptake in rat striatal synaptosomes (expressing DAT and SERT) and hippocampal synaptosomes (expressing SERT, but not DAT). Nonspecific [³H]serotonin uptake was defined as the amount of uptake remaining in the presence of fluoxetine (10 μM) or paroxetine (0.05 μM). In hippocampal synaptosomes, K_m and V_max values for [³H]serotonin uptake did not differ whether fluoxetine or paroxetine was used to define nonspecific uptake. However, in striatal synaptosomes, both K_m and V_max values for [³H]serotonin uptake were greater when fluoxetine, rather than paroxetine, was used to define nonspecific uptake. These data suggest that, at the concentrations employed, fluoxetine inhibits serotonin uptake at both DAT and SERT, whereas paroxetine only inhibits serotonin uptake at SERT. Thus, when DAT is inhibited by GBR 12909, kinetic parameters for serotonin uptake via SERT in striatum are not different from those obtained in hippocampus. These findings have important implications regarding the analysis of monoaminergic reuptake in brain regions exhibiting heterogeneous transporter expression.     

Lindane inhibition of [35S]TBPS binding to the GABAA receptor in rat brain.

The inhibition of [³⁵S]t-butylbicyclophosphorothionate ([³⁵S]TBPS) binding to the GABA_A receptor by the insecticide γ-hexachlorocyclohexane, lindane, was studied in several brain regions and using different membrane preparation methods, both in vitro and after dosing the animals with the chemical. In the latter studies, the amount of lindane remaining in the membrane suspensions used for binding assays was determined. In vitro data showed values of IC₅₀ from 150 to 1675 nM, varying in function of the membrane preparation method used. This may account for the discrepancies in IC₅₀ values found in the literature. IC₅₀ values within the range of 150–250 nM were determined using extensively washed membranes from several brain regions, so no evidence arose for brain regional differences in the affinity of lindane for the TBPS binding site. After different schedules of acute treatment with lindane, we found a manifest relationship between the extent of the observable inhibition of [³⁵S]TBPS binding and the lindane amount remaining in the membrane suspensions used for binding assays. This relationship was in good agreement with the in vitro data, so no support for an in vivo acute regulation of the binding site was obtained.     

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.     

Diazepam-insensitive GABAA receptors in rat cerebellum and thalamus

Three major populations of GABA_A receptor binding sites are present in cerebellar membranes: diazepam-sensitive [³H]Ro15-4513 binding sites, diazepam-insensitive [³H]Ro15-4513 binding sites and high-affinity [³H]muscimol binding sites. All three populations contain a β subunit as shown by immunoprecipitation with antibodies that recognize all β subunits. The β3 subtype of β subunit is contained in all three populations, but only a similar low fraction (< 20%) in each. Thus, the majority contain β subunits other than β3 (β2 and β1) and β3 subunits are not selectively associated with nor lacking in any of the three binding populations. Antibodies to the γ2 subunit precipitated similar fractions of [³H]Ro15-4513, [³H]flunitrazepam and [³H]muscimol binding sites, showing that γ2 subunits are present in high-affinity muscimol binding isoforms, as well as a significant fraction of the diazepam-insensitive [³H]Ro15-4513 binding sites. Under conditions that identify the 56 kDa 6 subunit on SDS-PAGE as the diazepam-insensitive site of [³H]Ro15-4513 binding in cerebellum, no polypeptide showing diazepam-insensitive binding of [³H]Ro15-4513 could be photoaffinity-labeled in rat thalamus. These results suggest that 4 subunits in the thalamus participate primarily in subunit combinations which bind muscimol but not any benzodiazepine site ligands.