Orexins/Hypocretins Acting at G<Subscript>i</Subscript> Protein-Coupled OX<Subscript>2</Subscript> Receptors Inhibit Cyclic AMP Synthesis in the Primary Neuronal Cultures

Orexins A and B are newly discovered neuropeptides with pleiotropic activity. They signal through two G protein-coupled receptors: OX₁ and OX₂. In this study, we examined the expression of orexin receptors and effects of the receptors’ activation on cyclic AMP formation in the primary neuronal cell cultures from rat cerebral cortex. Both types of orexin receptors were expressed in rat cortical neurons; the level of OX₂R was markedly higher compared to OX₁R. Orexin A (an agonist of OX₁R and OX₂R) and [Ala¹¹-D-Leu¹⁵]orexin B (a selective agonist of OX₂R) did not affect basal cyclic AMP formation in the primary neuronal cell cultures. Both peptides (0.001–1 μM) inhibited, in a concentration-dependent manner and IC₅₀ values in low nanomolar range, the increase in the nucleotide production evoked by forskolin (1 μM; a direct activator of adenylyl cyclase), pituitary adenylate cyclase-activating polypeptide (PACAP27; 0.1 μM), and vasoactive intestinal peptide (VIP; 3 μM). Effects of orexin A on forskolin-, PACAP27-, and VIP-stimulated cyclic AMP synthesis were blocked by TCS OX2 29 (a selective antagonist of OX₂R), and unaffected by SB 408124 (a selective antagonist of OX₁R). Pretreatment of neuronal cell cultures with pertussis toxin (PTX) abolished the inhibitory action of orexin A on forskolin- and PACAP-stimulated cyclic AMP accumulation. It is suggested that in cultured rat cortical neurons orexins, acting at OX₂ receptors coupled to PTX-sensitive G_i protein, inhibit cyclic AMP synthesis.     

Pharmacological characterization of orally active cholinesterase inhibitory activity of Prunus persica L. Batsch in rats

Prunus persica L. Batsch water extract (PPE) is a potent acetylcholinesterase (AChE) inhibitor screened for the treatment of Alzheimer's disease. The effects of oral administration of the PPE were examined with comparison of those of selective butyrylcholinesterase inhibitors of 9-amino-1,2,3,4-tetrahydroacridine hydrochloride (tacrine) and tetraidopropylpyrophosphoramide (iso-OMPA) and a selective AChE inhibitor, donepezil, on the cholinesterase activity in the brain and plasma of rats. After the sequential solvent fractionation of the methanol extract of P. persica L. Batsch, the highest inhibitory fraction was that of chloroform (75%). The concentration that was required for 50% enzyme inhibition (IC₅₀ value) was 5.6 μg/mL. for the chloroform fraction. Oral administration of PPE or tacrine caused a dose-dependent inhibition of brain and plasma cholinesterase activities. The ID₅₀ values of these compounds for brain cholinesterase activity were 2.7 g/kg and 8.9 mg/kg, respectively. On the other hand, the ID₅₀ values for plasma cholinesterase activity were 18.6 g/kg and 27.5 mg/kg, respectively. Thus, the ratios of the ID₅₀ (plasma<brain) were 6.0 and 3.1, respectively. These results suggest that orally administered PPE satisfactorily penetrates into the brain and inhibits cholinesterase there and that PPE is a potent inhibitor of brain cholinesterase in comparison with plasma cholinesterase in vivo.     

Effects of inhibition of fatty acid amide hydrolase vs. the anandamide membrane transporter on TRPV1-mediated calcium responses in adult DRG neurons; the role of CB receptors

The aim of the present study was to investigate the relationship between TRPV1 stimulation and endocannabinoid-driven CB(1) receptor-mediated inhibition of activity in adult rat dorsal root ganglion (DRG) neurons, a model of primary afferent nociceptors. Calcium-imaging studies were performed to compare the effects of the fatty acid amide hydrolase (FAAH) inhibitor URB597 (1 microm) vs. the anandamide (AEA) uptake inhibitor UCM707 (1 microm) on capsaicin (100 nm) and N-arachidonoyl dopamine (NADA; 1 microm)-evoked changes in intracellular calcium [Ca(2+)](i) in DRG neurons. The ability of the CB(1) receptor antagonist AM251 (1 microm) to modulate the effects of URB597 and UCM707 was also determined. Suprafusion of NADA and capsaicin evoked robust increases in [Ca(2+)](i) in DRG neurons (89 +/- 4% and 132 +/- 6% of the depolarizing KCl response, respectively). Co-incubation with URB597 significantly attenuated both NADA and capsaicin-evoked increases in [Ca(2+)](i) (39 +/- 3% and 79 +/- 4% of KCl response, respectively). Similarly, co-incubation with UCM707 significantly attenuated both NADA and capsaicin-evoked increases in [Ca(2+)](i) (59 +/- 7% and 72 +/- 4% of KCl response, respectively). The CB(1) receptor antagonist AM251 significantly attenuated the effects of URB597 on NADA-evoked increases in [Ca(2+)](i) but not the effects of URB597 on capsaicin-evoked increases in [Ca(2+)](i). By contrast, AM251 significantly attenuated the inhibitory effects of UCM707 on both NADA and capsaicin-evoked increases in [Ca(2+)](i.) These data suggest that transport of both NADA and capsaicin into DRG neurons and the subsequent activation of TRPV1 is partly governed by FAAH-dependent mechanisms as well as via the putative AEA membrane transporter.     

Normal thermoregulatory responses to 3‐iodothyronamine,trace amines and amphetamine‐like psychostimulants in trace amine associated receptor 1 knockout mice

3‐Iodothyronamine (T1AM) is a metabolite of thyroid hormone. It is an agonist at trace amine‐associated receptor 1 (TAAR1), a recently identified receptor involved in monoaminergic regulation and a potential novel therapeutic target. Here, T1AM was studied using rhesus monkey TAAR1 and/or human dopamine transporter (DAT) co‐transfected cells, and wild‐type (WT) and TAAR1 knock‐out (KO) mice. The IC₅₀ of T1AM competition for binding of the DAT‐specific radio‐ligand [³H]CFT was highly similar in DAT cells, WT striatal synaptosomes and KO striatal synaptosomes (0.72–0.81 μM). T1AM inhibition of 10 nM [³H]dopamine uptake (IC₅₀: WT, 1.4 ± 0.5 μM; KO, 1.2 ± 0.4 μM) or 50 nM [³H]serotonin uptake (IC₅₀: WT, 4.5 ± 0.6 μM; KO, 4.7 ± 1.1 μM) in WT and KO synaptosomes was also highly similar. Unlike other TAAR1 agonists that are DAT substrates, TAAR1 signaling in response to T1AM was not enhanced in the presence of DAT as determined by CRE‐luciferase assay. In vivo, T1AM induced robust hypothermia in WT and KO mice equivalently and dose dependently (maximum change degrees Celsius: 50 mg/kg at 60 min: WT ?6.0 ± 0.4, KO ?5.6 ± 1.0; and 25 mg/kg at 30 min: WT ?2.7 ± 0.4, KO ?3.0 ± 0.2). Other TAAR1 agonists including beta–phenylethylamine (β‐PEA), MDMA (3,4‐methylenedioxymethamphetamine) and methamphetamine also induced significant, time‐dependent thermoregulatory responses that were alike in WT and KO mice. Therefore, TAAR1 co‐expression does not alter T1AM binding to DAT in vitro nor T1AM inhibition of [³H]monoamine uptake ex vivo, and TAAR1 agonist‐induced thermoregulatory responses are TAAR1‐independent. Accordingly, TAAR1‐directed compounds will likely not affect thermoregulation nor are they likely to be cryogens. © 2010 Wiley‐Liss, Inc.     

Molecular cloning and functional expression of the mouse dopamine transporter

Summary. The full coding region of the murine dopamine transporter (mDAT) cDNA was cloned by PCR with a sense primer derived from the partial mDAT gene sequence and an antisense primer deduced from the rat dopamine transporter cDNA. The mDAT cDNA encodes a typical member of the family of Na⁺- and Cl−-dependent neurotransmitter transporters with 99.2; 93.4 and 85.4% amino acid identity to the rat, human and bovine DATs, respectively. Functional expression of the mDAT cDNA in transiently transfected human embryonic kidney (HEK293) cells exhibited the typical pharmacological features of a dopamine transporter. [³H]dopamine uptake through the mDAT was inhibited with high potency by GBR12909 (IC₅₀ = 5.2 nM) and not significantly affected by 100 nM desipramine. [³H]dopamine uptake also was inhibited through increasing concentrations of dopamine (IC₅₀ = 0.93 μM) or 1-methyl-4-phenylpyridinium (MPP⁺; IC₅₀ = 13.2 μM). Received May 17, 1999; accepted May 19, 1999     

Involvement of endocannabinoids in antidepressant and anti-compulsive effect of fluoxetine in mice

Endocannabinoid analogues exhibit antidepressant and anti-compulsive like effects similar to that of serotonin selective reuptake inhibitors (SSRIs) indicating a parallelism between the effects of serotonin and endocannabinoids. Therefore, the present study was designed to investigate the role of endocannabinoids in the antidepressant and anti-compulsive like effect of fluoxetine using mice model of forced swim test (FST) and marble-burying behavior (MBB). The results revealed that intracerebroventricular injections of endocannabinoid analogues, anandamide, a CB₁ agonist (AEA: 1-20 μg/mouse); AM404, an anandamide transport inhibitor (0.1-10 μg/mouse); and URB597, a fatty acid amide hydrolase inhibitor (0.05-10 μg/mouse) produced antidepressant-like effect dose-dependently, whereas influenced the MBB in a biphasic manner (produced a U-shaped dose-response curve). Fluoxetine (2.5-20 mg/kg, i.p.) dose dependently decreased the immobility time as well as burying behavior. Co-administration of sub-effective dose of fluoxetine (2.5 mg/kg, i.p.) potentiated the effect of sub-effective dose of AEA (0.5 μg/mouse, i.c.v.), AM404 (0.05 μg/mouse, i.c.v) or URB597 (0.01 μg/mouse, i.c.v) in both the paradigms. Interestingly, pretreatment with AM251, a CB₁ antagonist, blocked the effect of fluoxetine in FST and MBB at a dose (1 μg/mouse, i.c.v) that per se had no effect on either parameter. Similar effects were obtained with endocannabinoid analogues in AM251 pretreated mice. However, AM251 increased the burying behavior in MBB at a highest dose tested (5 μg/mouse). None of the treatments had any influence on locomotor activity. Thus, the study indicates an interaction between endocannabinoid and serotonergic system in regulation of depressive and compulsive-like behavior.     

Natural lipophilic inhibitors of mitochondrial complex I are candidate toxins for sporadic neurodegenerative tau pathologies

Annonacin, a natural lipophilic inhibitor of mitochondrial complex I has been implicated in the etiology of a sporadic neurodegenerative tauopathy in Guadeloupe. We therefore studied further compounds representing the broad biochemical spectrum of complex I inhibitors to which humans are potentially exposed.We determined their lipophilicity, their effect on complex I activity in submitochondrial particles, and their effect on cellular ATP levels, neuronal cell death and somatodendritic redistribution of phosphorylated tau protein (AD2 antibody against pS396/pS404-tau) in primary cultures of fetal rat striatum.The 24 compounds tested were lipophilic (logP range 0.9–8.5; exception: MPP⁺ logP = − 1.35) and potent complex I inhibitors (IC₅₀ range 0.9 nM–2.6 mM). They all decreased ATP levels (EC₅₀ range 1.9 nM–54.2 μM), induced neuronal cell death (EC₅₀ range 1.1 nM–54.5 μM) and caused the redistribution of AD2⁺ tau from axons to the cell body (EC₅ range 0.6 nM–33.3 μM). The potency of the compounds to inhibit complex I correlated with their potency to induce tau redistribution (r = 0.80, p < 0.001).In conclusion, we propose that the widely distributed lipophilic complex I inhibitors studied here might be implicated in the induction of tauopathies with global prevalence.     

A comparative optical aggregometry study of antiplatelet activity of taxanes from Taxus cuspidata

Platelets are highly reactive components of the circulatory system. The cytoskeleton of a platelet is an important structure for platelet aggregation as stimulated by several agonists. An anticancer agent, taxol, has been suggested to exert platelet anti-aggregating activity by stabilizing microtubules during the aggregation process. An activity-guided fractionation was performed with a methanol extract of the leaves and twigs of Taxus cuspidata to isolate taxanes with platelet anti-aggregating effects. Compounds 1 to 7 – taxinine (1), taxinine A (2), taxinine B (3), 2-deacetoxytaxinine B (4), taxacin (5), taxchinin B (6), and taxol (7) – were obtained as the antiplatelet components of this plant. These taxane compounds present the possibility of securing new antiplatelet compounds which differ from currently available antiplatelet agents in chemical structure and possibly in mechanisms of action. All compounds showed stronger inhibitory effects than acetylsalicylic acid (ASA) on platelet aggregation induced by arachidonic acid (AA) (IC₅₀: 14.4, 64.5, 35.5, 16.0, 21.9, 28.6 and 48.2 versus 63.0 μM) or U46619 (IC₅₀: 34.8, 24.9, 36.2, 35.0, 46.9, 71.9 and 68.7 versus 340 μM). Compounds 1, 3, 4 and 5, with a cinnamoyl group at the C₅ position, showed strong inhibitory effects against AA-induced aggregation compared to compound 2 (with an -OH group at C₅) or compounds with an oxetane ring at C₄,₅, such as compounds 6 and 7. All of the seven compounds were 5–13-fold more strongly inhibitory than ASA against U46619-induced aggregation.     

Monoamine oxidase inhibitory activities of novel 3,4-dihydroquinolin-(<Emphasis Type="Italic">1H</Emphasis>)-2-one derivatives

Summary Three 3,4-dihydroquinoline-(1H)-2-one derivatives were synthesized and their monoamine oxidase (MAO) inhibitory activities were evaluated. The calculated IC₅₀ values revealed that compound Q (N-amino-3,4-dihydroquinoline-(1H)-2-one), which carries a free amine group in the molecule, inhibited rat liver MAO-B competitively and reversibly suggesting that this relatively small compound may interact with the active site channel of the enzyme while the compounds QB (1-(benzlyden-amino)-3,4-dihydroquinoline-(1H)-2-one), PCN (2-(3-cyano-2-oxo-4-phenyl-2H-quinolin-1-yl-N-cyclohexyl-2-(4′-chlorophenyl) acetamide) and MG (tert-butyl-N-[cyclohexylcarbamoyl-(3-hydroxyphenyl)methyl]-N-(2-benzoylphenyl)-carbamate) inhibited rat liver MAO-B non-competitively and irreversibly, suggesting that these compounds may interact with another hydrophobic binding region outside of the active site of the enzyme.     

Characterization of the effects of reuptake and hydrolysis inhibition on interstitial endocannabinoid levels in the brain: an in vivo microdialysis study

The present experiments employed in vivo microdialysis to characterize the effects of commonly used endocannabinoid clearance inhibitors on basal and depolarization-induced alterations in interstitial endocannabinoid levels in the nucleus accumbens of rat brain. Compounds targeting the putative endocannabinoid transporter and hydrolytic enzymes (FAAH and MAGL) were compared. The transporter inhibitor AM404 modestly enhanced depolarization-induced increases in 2-arachidonoyl glycerol (2-AG) levels but did not alter levels of N-arachidonoyl-ethanolamide (anandamide, AEA). The transport inhibitor UCM707 did not alter dialysate levels of either endocannabinoid. The FAAH inhibitors URB597 and PF-3845 robustly increased AEA levels during depolarization without altering 2-AG levels. The MAGL inhibitor URB602 significantly enhanced depolarization-induced increases in 2-AG, but did not alter AEA levels. In contrast, the MAGL inhibitor JZL184 did not alter 2-AG or AEA levels under any condition tested. Finally, the dual FAAH/MAGL inhibitor JZL195 significantly enhanced depolarization-induced increases in both AEA and 2-AG levels. In contrast to the present observations in rats, prior work in mice has demonstrated a robust JZL184-induced enhancement of depolarization-induced increases in dialysate 2-AG. Thus, to further investigate species differences, additional tests with JZL184, PF-3845, and JZL195 were performed in mice. Consistent with prior reports, JZL184 significantly enhanced depolarization-induced increases in 2-AG without altering AEA levels. PF-3845 and JZL195 produced profiles in mouse dialysates comparable to those observed in rats. These findings confirm that interstitial endocannabinoid levels in the brain can be selectively manipulated by endocannabinoid clearance inhibitors. While PF-3845 and JZL195 produce similar effects in both rats and mice, substantial species differences in JZL184 efficacy are evident, which is consistent with previous studies.     

Taurine inhibition of metal-stimulated catecholamine oxidation

Taurine is an abundant amino acid found in mammalian tissues and it has been suggested to have cyto-protective functions. The aim of the present study was to determine if taurine had the potential to reduce oxidative stress associated with metal-stimulated catecholamine oxidation. Taurine and structural analogs of taurine were tested for their ability to inhibit metal-stimulated quinone formation from dopamine or L-dopa. Oxidative damage to proteins and lipids were also assessedin vitro and the effects of taurine were determined. Taurine (20 mM) was found to decrease significantly ferric iron (50–500 μM)- and manganese (10 μM)-stimulated L-dopa or dopamine oxidation. Taurine had no effect on zinc-induced dopamine oxidation and slightly potentiated copper- and NaIO₄-stimulated quinone formation. Ferric iron-stimulated lipid peroxidation was not affected by taurine (1–20 mM). Protein carbonyl formation induced by ferric iron (500 μM) and L-dopa (500 μM) was significantly reduced by 10 mM taurine. The cytotoxicity of L-dopa (250 μM) and ferric chloride (75 μM) to LLC-PK₁ cells was attenuated by 10 mM taurine or hypotaurine. Homotaurine alone stimulated L-dopa oxidation and potentiated the cytotoxic effects of ferric iron. Homotaurine was found to be cytotoxic when combined with L-dopa or L-dopa/iron. In contrast, hypotaurine inhibited quinone formation and protected LLC-PK₁ cells. These studies suggest that taurine may exhibit cytoprotective effects against the oxidation products of catecholamines by acting as a scavenger for free radicals and cytotoxic quinones.     

sPhospholipase A<Subscript>2</Subscript> is inhibited by anthocyanidins

Epidemiological studies suggest that nutritional antioxidants may reduce the incidence of neurodegenerative disorders and age-related cognitive decline. Specifically, protection against oxidative stress and inflammation has served as a rationale for promoting diets rich in vegetables and fruits. The present study addresses secretory phospholipase A₂ (sPLA₂) as a novel candidate effector of neuroprotection conferred by anthocyanins and anthocyanidins. Using a photometric assay, 15 compounds were screened for their ability to inhibit PLA₂. Of these, cyanidin, malvidin, peonidin, petunidin, and delphinidin achieved K _i values ≤18 μM, suggesting a modulatory role for berry polyphenols in phospholipid metabolism.     

Activation of 5-hydroxytryptamine1A receptors increases the affinity of galanin receptors in di- and telencephalic areas of the rat

Since galanin in vitro selectively increases theK_D value of 5-HT_1A receptors without altering the binding of 5-HT_1B or 5-HT₂ receptors, we have studied whether 5-HT_1A receptor activation in turn may affect galanin binding in the ventral di- and telencephalon and the substantia nigra of the rat. As analyzed by autoradiography, the binding of¹²⁵I-galanin was increased by about 55% in the presence of 3–30 nM of 8-OH-2-(di-npropylamino)-tetralin (DPAT) in the paraventricular thalamic nucleus, the nucleus reuniens and rhomboideus, the zona incerta, the medial and the lateral hypothalamus, and the medial and the lateral amygdaloid area, but not in the pars compacta of the substantia nigra, which lacks 5-HT_1A binding sites. DPAT (10 nM) reduced the IC₅₀ values of galanin at¹²⁵I-galanin binding sites by approximately 55% within all the analyzed di- and telencephalic regions. The overall increase inB_O values was50 ± 11%. Using the filter wipe technique in cryostat sections at Bregma -2.8 mm covering all the brain regions at this level, DPAT (10 nM) decreased the IC₅₀ values of galanin from21.6 ± 1.1nM (control) to15.5 ± 0.9nM, and increased theB_O values by19.4 ± 4.1%. In membrane preparations from the ventral di- and telencephalon, DPAT decreased the IC₅₀ values of galanin binding sites by20 ± 3% at 100 nM of DPAT. This effect could be completely blocked by the specific 5-HT_1A receptor antagonist 1-(2-methoxyphenyl)-4-[4-(2-pthalimido)butyl]piperazine. GTP (0.1 nM) produced a17 ± 5% increase in the IC₅₀ value of galanin and a23 ± 4% decrease in theB_O value of¹²⁵I_galanin binding sites. However, DPAT (100 nM) was still able to decrease the IC₅₀ values of galanin in the presence of GTP (-8 ± 3%;control-10 ± 3%). TheB_max value of¹²⁵I-galanin binding was not affected by DPAT. The increased affinity of galanin binding sites by DPAT seems to reflect a G-protein-independent intramembrane receptor-receptor interaction between 5-HT_1A and galanin receptors. This interaction may represent an intramembrane inhibitory feed-back mechanism of 5-HT_1A receptor sensitivity, and may be important both under normal conditions and in 5-HT-mediated mental disorders.     

Glutamate is not involved in the MPP+-induced dopamine overflow in the striatum of freely moving C57BL/6 mice

Summary. The role of glutamate in the N-methyl-4-phenyl-dihydropyridinium (MPP⁺) toxicity has been argued in the past decade. However, the effects of glutamate efflux and NMDA antagonist on MPP⁺-induced dopamine overflow have not been documented. To clarify this, we perfused MPP⁺ through a microdialysis probe in the striatum of freely moving mature C57BL/6 mice. The 60-min perfusion of 10 and 100 μM MPP⁺ strikingly increased dopamine levels to 28- and 93-fold of the basal values, respectively. In contrast, an administration of MPP⁺ did not induce marked glutamate release: the MPP⁺-perfusion slightly increased the glutamate level at 100 μM, but not at 10 μM. The addition of 100 μM (+)-MK-801 or 200 μM (±)-AP-7 to the perfusate did not attenuate MPP⁺-induced dopamine overflow. The extent of dopamine release only depended on the amount of MPP⁺ accumulation into the cells. These results indicated that, at least in the striatum, neither glutamate release nor the NMDA antagonist, including (+)-MK-801, could regulate MPP⁺-evoked dopamine overflow. Received November 7, 2000; accepted February 28, 2001     

Equol and other compounds from bovine urine as monoamine oxidase inhibitors

Summary Equol, its methylated derivative, and a carbazole, all isolated from bovine urine, are relatively potent inhibitors of monoamine oxidase with IC₅₀ values of 158, 28, and 16M respectively (using 83M tyramine as substrate). The probable dietary origin of these compounds suggests that natural monoamine oxidase inhibitors may be more widespread than had previously been suspected.     

Fencamfamine modulates sodium, potassium-ATPase through cyclic AMP and cyclic AMP-dependent protein kinase in rat striatum

Summary. Dopamine (DA) and fencamfamine (FCF) modulatory action on Na,K-ATPase and Mg-ATPase activity were evaluated in rat striatum. DA and FCF induced a decrease in Na,K-ATPase, without affecting Mg-ATPase activity. The effect of FCF was dose-dependent from 10 to 100 μM, with an IC₅₀ of 4.7 × 10⁻⁵ M. Furthermore, the effect of FCF (100 μM) increasing AMPc levels, but not GMPc, was nonadditive with that of DA (10 μM), which is consistent to a common site of action. The 8-bromo-cyclic AMP also induced a specific reduction in the Na,K-ATPase activity. The reduction of Na,K-ATPase induced by FCF (100 μM) was blocked by either SCH 23390 or sulpiride, which are D1 and D2 receptor antagonists. The decrease in striatal NA,K-ATPase activity induced by FCF was blocked by KT 5720, a selective inhibitor of cyclic AMP-dependent protein kinase (PKA), but not by KT 5823, a selective inhibitor of cyclic GMP-dependent protein kinase (PKG). Otherwise, KT 5720 or KT 5823 did not produce any change in Na,K-ATPase or Mg-ATPase activity. These data suggest that FCF reduces Na,K-ATPase activity through cyclic AMP-dependent changes in protein phosphorylation via a PKA mechanism. Accepted January 20, 1998; received June 2, 1997     

Endocannabinoids mediate anxiolytic-like effect of acetaminophen via CB1 receptors

Acetaminophen (Paracetamol), a most commonly used antipyretic/analgesic agent, is metabolized to AM404 (N-arachidonoylphenolamine) that inhibits uptake and degradation of anandamide which is reported to mediate the analgesic action of acetaminophen via CB1 receptor. AM404 and anandamide are also reported to produce anxiolytic-like behavior. In view of the implication of endocannabinoids in the effect of acetaminophen, we contemplated that acetaminophen may have anxiolytic-like effect. Therefore, this possibility was tested by observing the effects of various doses of acetaminophen in mice on anxiety-related indices of Vogel conflict test and social interaction test. The results from both the tests indicated that acetaminophen (50, 100, or 200 mg/kg, i.p.) or anandamide (10 or 20 µg/mouse, i.c.v.) dose dependently elicited anxiolytic-like effect, that was comparable to diazepam (2 mg/kg, i.p.). Moreover, co-administration of sub-effective dose of acetaminophen (25 mg/kg, i.p.) and anandamide (5 µg/mouse, i.c.v) produced similar anxiolytic effect. Further, pre-treatment with AM251 (a CB1 receptor antagonist; 1 mg/kg, i.p.) antagonized the effects of acetaminophen and anandamide with no per se effect at 1 mg/kg dose, while anxiogenic effect was evident at a higher dose (5 mg/kg, i.p.). None of the treatment/s was found to induce any antinociceptive or locomotor impairment effects. In conclusion, the findings suggested that acetaminophen (50, 100, or 200 mg/kg, i.p.) exhibited dose dependent anxiolytic effect in mice and probably involved endocannabinoid-mediated mechanism in its effect.     

-DOPA transport properties in an immortalised cell line of rat capillary cerebral endothelial cells, RBE 4

The present study aimed to determine the kinetics of -3,4-dihydroxyphenylalanine ( -DOPA) uptake in an immortalised cell line of rat capillary cerebral endothelial cells (clones RBE 4 and RBE 4B), to define the type of inhibition produced by -5-hydroxytryptophan ( -5-HTP), 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BHC) and N-(methylamino)-isobutyric acid (MeAlB) and its sodium dependence. Non-linear analysis of the saturation curves for -DOPA and -5-HTP revealed in RBE 4 cells K_m values (in μM) of 72 and 102 and in RBE 4B cells K_m values (in μM) of 60 and 118, respectively. IC₅₀ values for -5-HTP (RBE 4, 1026 μM; RBE 4B, 831 μM) obtained in the presence of a nearly saturating (250 μM) concentration of -DOPA were almost 5-fold those obtained when non-saturating (25 μM) concentrations of -DOPA were used. IC₅₀ values for BHC obtained in the presence of a nearly saturating (250 μM) concentration of -DOPA were also 6- to 5-fold those obtained when non-saturating (25 μM) concentrations of -DOPA were used. MeAlB (up to 2.5 mM) was found not to interfere with the uptake of -DOPA. In RBE 4 cells, V_max values for -DOPA uptake were identical in the absence and the presence of 150 μM -5-HTP or 150 μM BHC, but K_m values (μM) were significantly greater (P<0.05) when -DOPA uptake was studied in the presence of -5-HTP or BHC. Similar findings were observed when RBE 4B cells were used. Uptake of (250 μM) -DOPA in the absence of sodium in the incubation medium was similar to that observed in the presence of increasing concentrations of sodium (20 to 140 mM). It is concluded that RBE 4 and RBE 4B cells are endowed with the L-type amino acid transporter through which -DOPA and -5-HTP can be taken up, and suggested that this immortalised cell line of rat capillary cerebral endothelium might constitute an interesting in vitro model for the study of BBB mechanisms, namely those concerning solute and nutrient transfer across the brain capillary endothelium.     

Effect of metabotropic glutamate receptor activation on receptor-mediated cyclic AMP responses in primary cultures of rat striatal neurones

Co-activation of group I metabotropic glutamate (mGlu) receptors and adenosine receptors resulted in an augmented cyclic AMP response in primary cultures of rat striatal neurones. -glutamate and the selective group I agonist, (S)-dihydroxyphenylglycine (S-DHPG) evoked concentration-dependent potentiations of cyclic AMP accumulation stimulated by the adenosine receptor agonist, 5′-N-ethylcarboxamidoadenosine (NECA), with EC₅₀ values of 3.41±0.39 and 5.69±1.64 μM, respectively, and maximal augmentations of approximately 350% at concentrations of 100 μM. The S-DHPG potentiation was inhibited by group I mGlu receptor antagonists and a protein kinase C inhibitor, Ro 31-8220, implicating products of PI hydrolysis in this effect. Furthermore, -glutamate and S-DHPG stimulated PI hydrolysis in striatal neuronal cultures with similar EC₅₀ values to those observed for the augmentation of NECA cyclic AMP responses (5.19±1.18 and 3.78±1.42 μM, respectively). In situ hybridization and immunofluorescence techniques indicate that group I mGlu receptor-evoked potentiations are likely to be mediated via mGlu₅ receptors, which are expressed at high levels in these cultures. In contrast to cross-chopped slices of neonatal rat striatum, of equivalent age, the group II mGlu receptor agonist, (2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine (DCG-IV) was without effect on NECA- or forskolin-stimulated cyclic AMP responses in primary striatal neuronal cultures. This lack of effect might be due to a low level of expression of group II mGlu receptors in cultured striatal neurones.     

Interactions of dextromethorphan with theN-methyl-d-aspartate receptor-channel complex: single channel recordings

The actions of dextromethorphan (DXM) on the 50 pS conductance state of theN-methyl-d-aspartate (NMDA) receptor-operated channel were studied using outside-out patches obtained from cultured rat hippocampal pyramidal neurons. DXM (5–50 μM) had no effect on the amplitudes of unitary currents but caused concentration-dependent reductions in channel mean open times and the frequency of channel openings. Channel open probability was reduced in a concentration-dependent manner by DXM and was one-half of the control value at a DXM concentration of 6 μM, with the patch potential held at −60 mV. An IC₅₀ value of 4 μM was obtained for the reduction by DXM of NMDA-evoked rises in [Ca²⁺]_i in cultured rat hippocampal pyramidal neurons loaded with Fura-2. The results were consistent with drug block of the open NMDA channel with an onward (blocking) rate constant of 7.7 × 10⁶ M⁻¹ · s⁻¹ (at −60 mV). The estimated unblocking rate constant was about 10 s⁻¹, a value considerably higher compared to the off-rate constant found for dizocilpine block of the NMDA channel.