Co-agonism in drug-receptor interaction: illustrated by the NMDA receptors.

The co-agonism between glutamate and glycine at the NMDA receptor raises uncertainty about the estimation of the values of dissociation constants for agonists and antagonists and of efficacy for agonists. In this article, Mauro Corsi, Paolo Fina and David Trist discuss how to analyse the interaction of the two agonists with the NMDA receptor by applying an operational receptor model. Data simulation indicates that co-agonism can affect the potency as well as the efficacy of agonists. Moreover, the interaction of antagonists with the NMDA receptor can also be affected, leading to altered estimation of the antagonist dissociation constants.

NMDA NR1 subunit mRNA and glutamate NMDA-sensitive binding are differentially affected in the striatum and pre-frontal cortex of Parkinson's disease patients.

Changes in the levels of mRNA for the NR1 subunit of the glutamate NMDA receptor and in NMDA-sensitive glutamate binding were investigated in consecutive sections of the prefrontal cortex and striatum of control and Parkinson's disease (PD) post-mortem brain using in-situ hybridisation and receptor autoradiography. Both markers of NMDA receptors were found to be relatively unaffected when measured by microdensitometry in the prefrontal cortex of control and PD brains. At a cellular level, a subpopulation of small and medium neurons in the superficial layers of the prefrontal cortex of the PD group showed a decreased expression of NMDA NR1 mRNA, with the maximal decrease in cortical layer IV. In the striatum, levels of glutamate binding to the NMDA receptor detected by receptor autoradiodgraphy were significantly reduced in the PD group, while no change could be detected at a macroscopical level in NMDA NR1 mRNA expression. Consequently, we suggest that the important decrease in agonist binding to the NMDA receptor observed in this study in the caudate and putamen of PD brains, in the absence of any major change in NMDA NR1 mRNA levels might reflect the degeneration of pre-synaptic NMDA receptors located on nigro-striatal projections particularly affected by the disease. Small changes observed at a cellular level in subsets of neurons of both prefrontal cortex and striatum will be discussed at the light of neurochemical changes characteristics of PD.

Substituted indole-2-carboxylates as in vivo potent antagonists acting as the strychnine-insensitive glycine binding site.

A series of indole-2-carboxylates bearing suitable chains at the C-3 position of the indole nucleus was synthesized and evaluated in terms of in vitro affinity using [3H]glycine binding assay and in vivo potency by inhibition of convulsions induced by N-methyl-D-aspartate (NMDA) in mice. 3-[2-[(Phenylamino)carbonyl]ethenyl]-4,6-dichloroindole-2-carboxyl ic acid (8) was an antagonist at the strychnine-insensitive glycine binding site (noncompetitive inhibition of the binding of [3H]TCP, pA2 = 8.1) displaying nanomolar affinity for the glycine binding site (pKi = 8.5), coupled with high glutamate receptor selectivity (> 1000-fold relative to the affinity at the NMDA, AMPA, and kainate binding sites). This indole derivative inhibited convulsions induced by NMDA in mice, when administered by both iv and po routes (ED50 = 0.06 and 6 mg/kg, respectively). The effect of the substituents on the terminal phenyl ring of the C-3 side chain was investigated. QSAR analysis suggested that the pKi value decreases with lipophilicity and steric bulk of substituents and increases with the electron donor resonance effect of the groups present in the para position of the terminal phenyl ring. According to these results the terminal phenyl ring of the C-3 side chain should lie in a nonhydrophobic pocket of limited size, refining the proposed pharmacophore model of the glycine binding site associated with the NMDA receptor.

Synthesis and SAR of new 5-phenyl-3-ureido-1,5-benzodiazepines as cholecystokinin-B receptor antagonists.

A series of 5-phenyl-3-ureidobenzodiazepine-2,4-diones was synthesized and evaluated as cholecystokinin-B (CCK-B) receptor antagonists. Structure-activity relationship (SAR) studies revealed the importance of the N-1 substituent for potent and selective CCK-B affinity. Addition of substituents at the urea side chain provided in some cases more potent compounds. Moreover the introduction of bulky substituents such as adamantylmethyl at N-1 and resolution of the racemic ureas resulted in our lead compound GV150013.

Discrimination by benextramine between the NPY-Y1 receptor subtypes present in rabbit isolated vas deferens and saphenous vein.

1. In order to characterize the neuropeptide Y (NPY) Y1 receptors known to be present in rabbit isolated vas deferens and saphenous vein, the pharmacological activity of the selective NPY Y1 receptor agonists, [Leu31,Pro34] NPY and various other peptide agonists, together with the putative NPY antagonist, benextramine, were compared in the two tissues. 2. In rabbit isolated saphenous vein, cumulative dose-response curves to various NPY agonists were obtained. All the peptides tested caused contractions which developed quite slowly. The rank order of potency obtained was: PYY > NPY > [Leu31,Pro34] NPY = NPY2-36 > hPP >> NPY13-36 = NPY18-36. Incubation with benextramine (BXT) at 100 microM for 30 min irreversibly abolished the contractile response to [Leu31,Pro34] NPY but was ineffective against NPY18-36-induced contractions. 3. Cumulative dose-response curves to [Leu31,Pro34] NPY were performed in the same preparation before and after incubation with 100 microM BXT for 20 min in order to inactivate NPY Y1 receptors. The pKA (-logKA) estimation for [Leu31,Pro34] NPY was 7.60 +/- 0.30 using the operational model and 7.20 +/- 0.33 using the null method; the difference between the two methods was not statistically significant (P = 0.36). 4. Prostatic segments of rabbit vas deferens were electrically stimulated with single pulses. Immediately after stabilization of the contractile response, a cumulative dose-response curve to various NPY agonists was obtained in each tissue. The rank order of potency for twitch inhibition was: PYY> [Leu31,Pro34]NPY > NPY > hPP>NPY2- 36 >>NPY13-36>> NPY 18-36 which indicates the presence of a prejunctional NPY Y1 receptor. BXT at 100 microM incubated for 10 or 60 min did not antagonize the response to[Leu31,Pro34] NPY.5. We conclude that rabbit isolated saphenous vein contains a population of post-junctional NPY Y1 receptors irreversibly blocked by BXT, as well as a population of post-junctional NPY Y2 receptors,which are insensitive to BXT. In contrast, the rabbit isolated vas deferens express a pre-junctional NPYY1 receptor subtype which is not blocked by BXT. Tetramine disulphides such as BXT could be useful tools in classifying NPY receptors.

Evidence for the presence of both pre- and postjunctional P2-purinoceptor subtypes in human isolated urinary bladder.

1. In order to characterize P2-purinoceptor(s) in human urinary bladder the contractile effects of ATP and its slowly-hydrolyzable analogues alpha, beta-methylene ATP (alpha, beta-MeATP) and beta, gamma-methylene ATP (beta, gamma-MeATP) were investigated on human detrusor strips taken from patients undergoing cystectomy for bladder carcinoma. 2. Serial concentration-response curves (SCRC) for ATP, alpha, beta-MeATP and beta, gamma-MeATP were constructed with an interval of 25 min between two successive doses to avoid tachyphylaxis. ATP (10 microM-10 mM) induced a phasic contraction, which was very rapid in onset. The dose-response curve to ATP appeared not to be monophasic: at the lower concentrations (10-300 microM) the curve was shallow, whilst at high concentrations (1-10 mM) the curve was steeper. The magnitude of the response obtained at the highest concentration tested (10 mM) was only 21.1 +/- 2.8% (mean +/- s.e. mean; n = 4) of the KCl (100 mM)-induced contraction. 3. alpha, beta-MeATP (0.3 microM-1 mM) and beta, gamma-MeATP (10 microM-1 mM) elicited a phasic contraction with a time course similar to that exhibited by ATP. The magnitude of the response obtained at the highest concentration tested (1 mM) was 70.3 +/- 6.3% for alpha, beta-MeATP (n = 10) and 27.9 +/- 4.5% for beta, gamma-MeATP (n = 8) of KCl (100 mM)-induced contraction. The rank order of potency was alpha, beta-MeATP > beta, gamma-MeATP > ATP. A plateau of response could not be achieved by any of these agonists. 4. The P2-purinoceptor antagonist, suramin (10-300 microM), dose-dependently antagonized only the lower part of alpha,beta-MeATP dose-response curve. Data were analysed in terms of dose-ratio estimated at two levels of response (10% and 35% of KC1 100 mM-induced contraction). At 10% of KCl response the Schild plot slope was 0.98 and the estimated pKB was 5.85, whereas using the dose-ratio at the 35% level of the KCl response, the Schild plot was not linear suggesting an interaction of alpha,beta-MeATP with a heterogeneous receptor population.5. The putative P2-purinoceptor antagonist, Coomassie Brilliant Blue G (CB-G) at 0.3 and 1 l micro M(n = 5), shifted to the left the alpha,beta-MeATP SCRC. The response at the highest concentration of agonist was potentiated, being equal to 78.8 +/- 11.7% of the KCl (100 mM) response (n = 5). CB-G at 0.3 microM also shifted to the left the beta,upsilon-MeATP SCRC and significantly potentiated the response at 1 mM up to 46.3 +/- 5.6% of KCl 100 mM response (n = 4).6. Pretreatment with terodotoxin (TTX) at 1 microM shifted to the left the alpha,beta-MeATP SCRC but the response to the highest concentration of the agonist was not potentiated, being 73.6 +/- 9.9% of the KCl(100 mM) response (n = 5). TTX (1 micro M) shifted to the left the beta,upsilon-MeATP SCRC and significantly potentiated the response at 1 mM (61.6 +/- 3.1% of KCl response; n = 4).7. The NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) at 100 micro M did not modify the SCRC to either alpha, beta or beta,upsilon-MeATP.8. We conclude that in human detrusor muscle there is a heterogeneity of purinoceptors. The complex antagonism exhibited by suramin suggests the presence not only of Ph-purinoceptors but also of another contractile P2-purinoceptor subtype insensitive to suramin. Moreover, the activity of CB-G and TTX seems to support the existence of a prejunctional P2-purinoceptor subtype inducing the release of one or more inhibitor neurotransmitters.

The antagonism by BW A868C of PGD2 and BW245C activation of human platelet adenylate cyclase.

1. In glycerol-lysed human platelets, prostaglandin D2 (PGD2) and the hydantoin BW245C both activate adenylate cyclase in a biphasic manner. These activations are qualitatively different from those of carbacyclin, iloprost and prostaglandin E2 (PGE2) whose E/[A] curves can be adequately described by rectangular hyperbolae. 2. Prostaglandin E1 (PGE1) had E/[A] curves of slope significantly lower than that expected for a rectangular hyperbolae. 2. Prostaglandin E1 (PGE1) had E/[A] curves of slope significantly lower than that expected for a rectangular hyperbola. 3. The selective PGD2 antagonist BW A868C shifts the first phase of the PGD2 and BW245C E/[A] curves but has no effect on the second phase. 4. Applying a two-receptor model enables a pKB to be derived for BW A868C of 9.11. 5. BW A868C has no effect on carbacyclin, iloprost, prostacyclin, PGE1 and PGE2 at a concentration 1,000 fold that of its KB against PGD2 and BW245C. 6. These results indicate that PGD2 and BW245C are capable of activating adenylate cyclase in human platelets through the DP-receptor and by another mechanism as yet uncharacterized.

Failure of the putative neuropeptide Y antagonists, benextramine and PYX-2, to inhibit Y2 receptors in rat isolated prostatic vas deferens.

1. The pharmacological activity of neuropeptide Y (NPY) and some analogues in inhibiting the twitch contractions induced by electrical stimulation (single pulses at 25 V, 0.15 Hz, 1 ms) in the prostatic portion of the rat isolated vas deferens was investigated. The rank order of agonist potency was: PYY > NPY2-36 > NPY >> NPY13-36 >> NPY18-36 >> [Leu31,Pro34]NPY = hPP, which is consistent with the activation of a Y2 receptor. 2. The putative Y1 and Y2 antagonist, benextramine (BXT), incubated at 100 microM for 10 or 60 min, was ineffective against PYY-induced inhibition of the twitch response, suggesting that the prejunctional Y2 receptor in this tissue is different from the postjunctional one reported in the literature to be sensitive to BXT blockade. 3. The putative NPY antagonist, PYX-2, incubated at 1 microM for 20 min, was completely ineffective in antagonizing PYY-induced inhibition of twitches. 4. The twitch response was totally inhibited by suramin (100 microM) but was little affected by prazosin (1 microM). Furthermore, NPY was without effect on the dose-response curve to ATP in resting conditions. Taken together, these results suggest that in our paradigm, NPY inhibits the release of a purinergic neurotransmitter which mediates contraction of the prostatic portion of the rat vas deferens.

Regionally different N-methyl-D-aspartate receptors distinguished by ligand binding and quantitative autoradiography of [3H]-CGP 39653 in rat brain.

1. Binding of D,L-(E)-2-amino-4-[3H]-propyl-5-phosphono-3-pentenoic acid ([3H]-CGP 39653), a high affinity, selective antagonist at the glutamate site of the N-methyl-D-aspartate (NMDA) receptor, was investigated in rat brain by means of receptor binding and quantitative autoradiography techniques. 2. [3H]-CGP 39653 interacted with striatal and cerebellar membranes in a saturable manner and to a single binding site, with KD values of 15.5 nM and 10.0 nM and receptor binding densities (Bmax values) of 3.1 and 0.5 pmol mg-1 protein, respectively. These KD values were not significantly different from that previously reported in the cerebral cortex (10.7 nM). 3. Displacement analyses of [3H]-CGP 39653 in striatum and cerebellum, performed with L-glutamic acid, 3-((+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) and glycine showed a pharmacological profile similar to that reported in the cerebral cortex. L-Glutamic acid and CPP produced complete displacement of specific binding with Ki values not significantly different from the cerebral cortex. Glycine inhibited [3H]CGP 39653 binding with shallow, biphasic curves, characterized by a high and a low affinity component. Furthermore, glycine discriminated between these regions (P < 0.005, one-way ANOVA), since the apparent Ki of the high affinity component of the glycine inhibition curve (KiH) was significantly lower (Fisher's protected LSD) in the striatum than the cortex (33 nM and 104 nM, respectively). 4. Regional binding of [3H]-CGP 39653 to horizontal sections of rat brain revealed a heterogeneous distribution of binding sites, similar to that reported for other radiolabelled antagonists at the NMDA site (D-2-[3H]-amino-5-phosphonopentanoic acid ([3H]-D-AP5) and [3H]-CPP). High values of binding were detected in the hippocampal formation, cerebral cortex and thalamus, with low levels in striatum and cerebellum. 5. [3H]-CGP 39653 binding was inhibited by increasing concentrations of L-glutamic acid, CPP and glycine. L-Glutamic acid and CPP completely displaced specific binding in all regions tested, with similar IC50 values throughout. Similarly, glycine was able to inhibit the binding in all areas considered: 10 microM and 1 mM glycine reduced the binding to 80% and 65% of control (average between areas) respectively. The percentage of specific [3H]-CGP 39653 binding inhibited by 1 mM glycine varied among regions (P < 0.05, two-ways ANOVA). Multiple comparison, performed by Fisher's protected LSD method, showed that the inhibition was lower in striatum (72% of control), with respect to cortex (66% of control) and hippocampal formation (58% of control). 6. The inhibitory action of 10 microM glycine was reversed by 100 microM 7-chloro-kynurenic acid (7-CKA), a competitive antagonist of the glycine site of the NMDA receptor channel complex, in all areas tested. Moreover, reversal by 7-CKA was not the same in all regions (P < 0.05, two-ways ANOVA). In fact, in the presence of 10 microM glycine and 100 microM 7-KCA, specific [3H]-CGP 39653 binding in the striatum was 131% of control, which was significantly greater (Fisher's protected LSD) than binding in the hippocampus and the thalamus (104% and 112% of control, respectively). 7. These results demonstrate that [3H]-CGP 39653 binding can be inhibited by glycine in rat brain regions containing NMDA receptors; moreover, they suggest the existence of regionally distinct NMDA receptor subtypes with a different allosteric mechanism of [3H]-CGP 39653 binding modulation through the associated glycine site.

Pharmacological characterization of thromboxane and prostanoid receptors in human isolated urinary bladder.

1. Cumulative concentration-response curves (CRC) to prostaglandin E1 (PGE1), PGE2, PGD2 and PGF2alpha (0.01-30 microM) and to the thromboxane A2 (TXA2) receptor agonist U-46619 (0.01-30 microM) were constructed in human isolated detrusor muscle strips both in basal conditions and during electrical field stimulation. 2. All the agonists tested contracted the detrusor muscle. The rank order of agonist potency was: PGF2alpha > U-46619 > PGE2 whereas weak contractile responses were obtained with PGD2 and PGE1. Any of the agonists tested was able to induce a clear plateau of response even at 30 microM. 3. The selective TXA2 antagonist, GR 32191B (vapiprost), antagonized U-46619-induced contractions with an apparent pK(B) value of 8.27+/-0.12 (n = 4 for each antagonist concentration). GR 32191B (0.3 microM) did not antagonize the contractile responses to PGF2alpha and it was a non-surmountable antagonist of PGE2 (apparent pK(B) of 7.09+/-0.04; n = 5). The EP receptor antagonist AH 6809 at 10 microM shifted to the right the CRC to U-46619 (apparent pK(B) value of 5.88+/-0.04; n = 4). 4. Electrical field stimulation (20 Hz, 70 V, pulse width 0.1 ms, trains of 5 s every 60 s) elicited contractions fully sensitive to TTX (0.3 microM) and atropine (1 microM). U-46619 (0.01-3 microM) potentiated the twitch contraction in a dose-dependent manner and this effect was competitively antagonized by GR 32191B with an estimated pK(B) of 8.54+/-0.14 (n = 4 for each antagonist concentration). PGF2alpha in the range 0.01-10 microM (n = 7), but not PGE2 and PGE1 (n = 3 for each), also potentiated the twitch contraction of detrusor muscle strips (23.5+/-0.3% of KCl 100 mM-induced contraction) but this potentiation was unaffected by 0.3 microM GR 32191B (n = 5). 5. Cumulative additions of U-46619 (0.01-30 microM) were without effect on contractions induced by direct smooth muscle excitation (20 Hz, 40 V, 6 ms pulse width, trains of 2 s every 60 s, in the presence of TTX 1 microM; n = 3). Moreover, pretreatment of the tissue with 0.3 microM U-46619 did not potentiate the smooth muscle response to 7 microM bethanecol (n = 2). 6. We concluded that TXA2 can induce direct contraction of human isolated urinary bladder through the classical TXA2 receptor. Prostanoid receptors, fully activated by PGE2 and PGF2alpha are also present. All these receptors are probably located post-junctionally. The rank order of agonist potency and the fact that GR 32191B, but not AH6809, antagonized responses to PGE2 seem to indicate the presence of a new EP receptor subtype. Moreover, we suggest the presence of prejunctional TXA2 and FP receptors, potentiating acetylcholine release from cholinergic nerve terminals.

[3H]MK-801 binding and the mRNA for the NMDAR1 subunit of the NMDA receptor are differentially distributed in human and rat forebrain.

The distributions of [3H]MK-801 binding and the NMDA NR1 subunit mRNA were studied using receptor autoradiography and in-situ hybridization in rat and human brain whole-hemisphere coronal sections. Receptor protein detected by radioligand autoradiography and the mRNA for the key subunit of the receptor presented similar distributions in the forebrain, with a few areas showing an imbalance between the levels of mRNA and receptor protein. Human frontal cortex showed a relative abundance of NMDAR1 mRNA as compared to [3H]MK-801 binding. The same area in rat brain did not show any difference in the two distributions. In comparison, the rat claustrum presented a relative excess of NMDAR1 mRNA which was not detected in human sections. Human caudate nucleus exhibited relatively high levels of [3H]MK-801 binding that were unmatched in rat caudate. The hippocampi of either species presented similar levels of [3H]MK-801 binding and NMDAR1 mRNA, but when the two signals were measured in specific subfields of the hippocampal formation, the differential distribution of the two signals reflected the anatomy of hippocampal connections assuming a preferential dendritic distribution for MK-801 binding. Interestingly, rat and human hippocampi also showed some important species-dependent difference in the relative distribution of the receptor protein and mRNA. The data presented show an overall good correlation between the mRNA for the key subunit of the NMDA receptor and the functional receptor detected with radioligand binding and highlight the presence of local differences in their ratio. This may reflect different splicing of the mRNA for the NMDAR1 subunit in specific brain areas of rat and human. The species-dependent differences in the relative distribution of the mRNA for the key subunit of the NMDA receptor and that of a marker of functional receptors also highlights important differences in the NMDA function in rat and human brain.

Protective effect of GV150526A on the glutamate-induced changes in basal and electrically-stimulated cytosolic Ca++ in primary cultured cerebral cortical cells.

Glutamate-induced changes in intracellular free Ca++ concentration ([Ca++]i) were recorded in resting and electrically-stimulated primary cultures of rat cerebral cortical cells, employing the Ca++ indicator Fura 2. A brief (10 min) exposure to glutamate led to a concentration-dependent basal [Ca++]i increase, measured 30 min after glutamate removal. In order to unmask more subtle modifications in [Ca++]i movements associated with neurosecretion, the glutamate effect was also studied in electrically-stimulated cells. The application of trains (10 s) of electrical pulses (intensity 30 mA, duration 1 ms) induced frequency-related Na+- and Ca++-dependent [Ca++]i transients. A 5 min treatment with 50 microM glutamate reduced to 48% the electrically-evoked [Ca++]i transients, evaluated 30 min after glutamate challenge. The neuroprotective effect of sodium 4,6-dichloro-3-[(E)-3-(N-phenyl)propenamide]indole-2-carboxylate (GV150526A), a new indole derivative with high affinity and selectivity for the glycine site of the NMDA receptor-channel complex, was compared with that of DL-2-amino-5-phosphonopentanoic acid (AP5), ifenprodil, 7-chlorokynurenic acid and 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)-quinoxaline (NBQX) on glutamate-induced [Ca++]i changes in resting and electrically-stimulated cells. In both experimental conditions, GV150526A showed to be the most potent compound. Moreover, GV150526A and 7-chlorokynurenic acid were 2-3 times more active in stimulated neurons than in resting neurons, indicating a major involvement of the glycine site in the protection of the cells kept in an active state.

Further studies on the effects of selective neurokinin agonists upon the activation of micturition reflex in rats. Evidence for a dual NK-1 receptor mediated excitatory and inhibitory activity.

The ability of SP and some selective agonists for NK-1, NK-2 and NK-3 receptor subtypes to interfere with the micturition reflex after intra-arterial (i.a.) or intracerebroventricular (i.c.v.) administration was investigated in the urethane anaesthetized rat. When administered i.a. SP, the selective NK-1 agonist GR 73632 and the selective NK-2 agonists GR 64349 were equipotent to activate micturition reflex, both the tonic or rhythmic bladder contractions. GR 73632 but not GR 64349-induced activation of micturition reflex was antagonized in a dose-dependent manner by the selective NK-1 antagonist GR 82334. After i.c.v. administration SP, GR 73632 and the selective NK-1 agonist [Sar9,Met(0(2))11]-SP but not GR 64349 inhibited saline-induced activation of rhythmic bladder contractions; the order of potency was GR 73632 > [Sar9,Met(0(2))11]SP >> SP. Also the inhibitory effect of GR 73632 was dose-dependently affected by GR 82334. In the two models the selective NK-3 agonist senktide both after i.a. or i.c.v. administration induced neither excitatory or inhibitory activity. These findings suggest that neurokinins activate at the peripheral level the micturition reflex by an interaction at NK-1 and NK-2 receptor subtypes. In addition, NK-1 receptors appear to modulate, at the central level, the inhibition of the micturition reflex.

Simultaneous, selective detection of catecholaminergic and indolaminergic signals using cyclic voltammetry with treated micro-sensor.

Selective and simultaneous voltammetric analysis of catechols and indoles in vivo and in vitro has until now been feasible only by means of 'slow' scanning methods (scan speed in tens of seconds) such as differential pulse (DPV) and differential normal pulse voltammetry in conjunction with electrically and/or chemically treated carbon-fiber micro-electrodes (mCFE). Faster electrochemical techniques, such as chronoamperometry and cyclic voltammetry (CV), allow more rapid (seconds or fractions of a second) and frequent measurements of these chemicals. However, these methods show poor sensitivity and selectivity in the presence of different electroactive compounds with similar oxidation potentials. In order to analyze whether the lack of sensitivity and selectivity of the fast voltammetric methods results from the rapidity of the measurement or from the use of untreated sensors, the methods of CV (scan speed: 1000 mV/s) and DPV (scan speed: 10 mV/s) have been applied with either untreated or electrically treated mCFE to analyze the in vitro oxidation potential and current values of DA and 5-HT. When associated with untreated mCFE, neither method was able to separate and selectively detect the two compounds dissolved together in an inert vehicle; the voltammogram recorded resulted in a single broad oxidation signal. In contrast, when these techniques were performed with electrically treated mCFE, oxidation signals for DA (peak A) and 5-HT (peak B) were monitored simultaneously at approximately + 65 mV and + 240 mV, with DPV respectively, and at + 120 mV and + 300 mV with CV, respectively. Additionally, CV with treated mCFE on anesthetized rats, simultaneously monitored two striatal signals at approximately + 100 mV and + 300 mV. The oxidation values (Em) and current levels (nA) of these peaks remained stable during control recordings. The current levels were selectively increased by peripheral injection of fluphenazine (DA antagonist) or of 5-hydroxytryptophan (precursor of serotonin). The chemical nature of these two peaks may therefore be considered catecholaminergic and indolaminergic, respectively. Hence, this report provides the first evidence for the feasibility of concomitant in vitro analysis of DA and 5-HT using a rapid scanning method such as CV. In addition, the values of current level (nA) obtained with CV-mCFE for DA and 5-HT are comparable to those monitored with DPV-mCFE, supporting the view that treatment of the sensor is a key point for increasing the selectivity and the sensitivity of these voltammetric techniques. The feasibility of using CV with electrically treated mCFE for fast in vivo analysis of catechol and indole activities is also demonstrated.

Carbon fibre micro-electrodes for concomitant in vivo electrophysiological and voltammetric measurements: no reciprocal influences.

Differential pulse voltammetry and more recently cyclic voltammetry have been successfully used to monitor basal levels of endogenous chemicals by means of treated carbon fibre microbiosensors inserted in specific brain regions. In this study, feasibility of concomitant in vivo recordings of stable electrophysiological signals and basal ascorbate, catecholaminergic and indolaminergic voltammetric peaks at the same cerebral site by means of a single electrically treated carbon fibre micro electrode (microbiosensor) is presented. The results indicate that these two independent techniques can be combined in vivo at a single electrode, and that voltammetric measurements of unstimulated levels of extracellular compounds do not alter concomitant basal cell firing for a period long enough (more than 6 h) to allow pharmacological manipulations.

N-terminal splice variants of the NMDAR1 glutamate receptor subunit: differential expression in human and monkey brain.

The distribution of the mRNA coding for the two N-terminal splice variants of the N-methyl-D-aspartate receptor 1 (NMDAR1) subunit of the glutamate NMDA receptor was studied on whole-hemisphere human and macaca fascicularis brain sections by in-situ hybridisation. Synthetic oligonucleotides directed against NMDAR1a and NMDAR1b variants showed a specific distribution that was similar in human and monkey brain, with the NMDAR1a isoform present in the majority of the NMDA receptors, and the NMDAR1b variant present at high levels only in the cortex and dentate gyrus of the hippocampus. The distribution of the mRNAs for the NMDAR1pan and NMDAR1a subunit reported in this study support previous findings in rodent brain, while the restricted distribution of the NMDAR1b variant found in human and monkey suggests some important differences in the composition of the NMDA receptor in rodents and primates.

Inhibition of lymphocyte transformation by mepacrine and chloroquine.

Mepacrine inhibited uptake and the incorporation of leucine, thymidine and uridine into acid-insoluble material in human lymphocytes stimulated by phytohaemagglutinin (PHA) in vitro. The IC50 for each uptake was of the order of 2 microM. Mepacrine was inhibitory if applied at any time up to 48 h after PHA. The inhibition differed from that produced by colchicine and prostaglandin E1. The dose-response curve was steep, nearly all incorporation being inhibited by 2 x IC50. Chloroquine also had a steep dose-response curve, was about one-fifth as potent as mepacrine and was maximally effective in the first 6 h after PHA.

Inhibition of human lymphocyte transformation by two aryloxyalkylamidines.

The incorporation of [3H]thymidine into human lymphocytes stimulated by phytohaemagglutinin (PHA) was inhibited by anilino-N-2-m-chlorophenoxypropylacetamidine (501C) and xylamidine. These amidines antagonize 5-HT, but 5-HT did not alter [3H]thymidine incorporation. 501C inhibited PHA-induced lymphocyte transformation as observed by [3H]thymidine incorporation, [3H]uridine incorporation, [3H]leucine incorporation, DNA content, potassium content, and histological examination. 501C also inhibited increased [3H]thymidine incorporation in human mixed lymphocyte cultures. The IC50 of 501C for inhibition of these processes lay between 4 and 8 microM. When added late in culture (after 6-8 h) 501C was less effective. Possible mechanisms by which 501C inhibits transformation are discussed.

The uptake of mepacrine by horse polymorphonuclear leucocytes in vitro.

The uptake of mepacrine by isolated horse polymorphonuclear leucocytes (PMN) was measured using spectrophotofluorimetry. Two phases of uptake were observed, the first, rapid fraction, essentially complete by 10 min, and a second, slow fraction, which was still proceeding after 60 min. The appearance of mepacrine within the PMN was also visualized by fluorescence microscopy. Discrete yellow points of fluorescence were observed in the cytoplasm of PMN within 30 s. These discrete points corresponded both in size and number to the PMN granules. After 5 min, the nuclei showed faint fluorescence which increased in intensity with time. It was concluded that the accumulation of mepacrine by horse PMN consists of at least two phases, an initial rapid component which represents uptake of the drug through the plasma membrane into the cytoplasmic granules, and a subsequent slower component, possibly representing nuclear binding.

The accumulation of an aryloxylkylamidine (501C) and 5-hydroxytryptamine in human polymorphonuclear leucocytes: a quantitative electron microscopy study.

The uptake of anilino-N-2-m-chlorophenoxypropylacetamidine (501C) and 5-hydroxytryptamine (5-HT), in human polymorphonuclear leucocytes (PMN) was investigated by quantitative ultrastructural autoradiography. The major concentration of both drugs was in the cytoplasmic granules, 71.5 +/- 6.8% for [3H] 501C, and 68.75 +/- 6.1% for [3H]5-HT. Lesser quantities of both drugs were associated with the nucleus; 18.1 +/- 4.8% for [3H] 501C and 23.0 +/- 4.2% for [3H] 5-HT. Only small amounts of activity were recorded at other sites. The data suggests that there may be common binding sites for 501C and 5-HT in PMN. Furthermore the concentration of 501C in PMN granules may also account for the damaging effect of high concentrations of the drug on PMN.