Atypical” neuromodulatory profile of glutapyrone, a representative of a novel `class' of amino...

European Neuropsychopharmacology 8 (1998) 329–347

‘‘Atypical’’ neuromodulatory profile of glutapyrone, a representative of anovel ‘class’ of amino acid-containing dipeptide-mimicking 1,4-dihydropyridine (DHP) compounds: in vitro and in vivo studies

a ,b , a a a a*Ilga Misane , Vija Klusa , Maija Dambrova , Skaidrite Germane , Gunars Duburs ,a b b¨Egils Bisenieks , Roberto Rimondini , Sven Ove Ogren

aLaboratory of Pharmacology and Laboratory of Membrane Active Compounds and b-diketones, Latvian Institute of Organic Synthesis,Aizkraukles St. 21, Riga LV-1006, Latvia

bDivision of Cellular and Molecular Neurochemistry, Department of Neuroscience, Karolinska Institute, S-171 77 Stockholm, Sweden

Received 18 September 1997; accepted 9 November 1997

Abstract

Glutapyrone, a disodium salt of 2-(2,6-dimethyl-3,5-diethoxycarbonyl-1,4-dihydropyridine-4-carboxamido)-glutaric acid, is a repre-sentative of a novel ‘class’ of amino acid-containing 1,4-dihydropyridine (DHP) compounds developed at the Latvian Institute of OrganicSynthesis, Riga, Latvia. Conceptually, the glutapyrone molecule can be regarded as a dipeptide-mimicking structure formed by the ‘‘free’’amino acid (glutamate) moiety and ‘‘crypto’’ (built into the DHP cycle) amino acid (‘‘GABA’’) elements. Both of these amino acids arejoined by the peptide bond. This compound unlike classical DHPs lacks calcium antagonistic or agonistic properties. Our previous studiesrevealed a profound and long-term anticonvulsant, stress-protective and neurodeficit-preventive activities of glutapyrone. In view ofstructural properties the role of glutamatergic mechanisms in the mediation of central effects of glutapyrone was considered. In the present

3 3study glutapyrone at the concentration range of 1 mM–1 mM failed to effect both NMDA ([ H]TCP) and non-NMDA ([ H]KA and3[ H]AMPA) receptor ligand binding in the rat cortical membranes in vitro. The compound markedly enhanced motor hyperactivity

induced by the NMDA antagonist PCP and the dopamine releasing compound D-amphetamine in the rats. Glutapyrone displayed activityin a variety of animal models relevant for affective /depressive disorders in humans i.e. reserpine-induced ptosis and hypothermia, forcedswimming test and open field test. These data indicate that the unusually ‘‘broad’’ pharmacological spectrum of glutapyrone might involveconcomitant actions on multiple neurotransmitter systems, particularly, GABA-ergic and the catecholamines. It is discussed whether thesefunctional properties are secondary to action on intracellular events, predominantly, G protein-related since glutapyrone appears to lackdirect interactions with a number of receptors including ionotropic glutamate and GABA /Bzd receptors. 1998 Elsevier ScienceA

B.V. /ECNP. All rights reserved.

Keywords: Glutapyrone; 1,4-Dihydropyridines; Glutamate; GABA; Dipeptides; Receptors; Biogenic amine transporters; CNS-targetingdrugs; G proteins

1. Introduction cardiovascular regulatory profile (Morad et al., 1988) aswell as a variety of CNS-effects such as anticonvulsant /

Since the early 1980’s the presence of a dihydropyridine antiepileptic (Czuczwar et al., 1990a,b; Dolin et al., 1988;(DHP) ring in the structures of 1,4 dihydropyridine Karler et al., 1991), antinociceptive properties (Martin etderivatives (DHPs) has been regarded as a prerequisite for al., 1996) and activity in animal models of anxiety /depres-

21calcium (Ca ) channel modulating properties. The di- sion (Czyrak et al., 1990; Pucilowski, 1992; Tazi et al.,verse in vivo actions of the DHPs, i.e. their well-known 1991; Viveros et al., 1996) have been attributed to either

potentiation (Bay K 8644) (Schramm et al., 1983) or*Corresponding author. Tel.: 146-8-728-7071; fax: 146-8-302875. inhibition (nitrendipine, nimodipine, and nifedipine)

0924-977X/98/$ – see front matter 1998 Elsevier Science B.V. /ECNP. All rights reserved.PI I : S0924-977X( 97 )00097-7

330 I. Misane et al. / European Neuropsychopharmacology 8 (1998) 329 –347

(Braunwald, 1982; Cavero and Spedding, 1983; Triggle, several years, this approach has led to development of1990) of calcium influx mediated by the high threshold novel type of 1,4-dihydropyridine derivatives at the Lat-L-type voltage-sensitive calcium channels (VSCC). How- vian Institute of Organic Synthesis, Latvia, Riga. The workever, recent advances in drug development have led to has been focused on the synthesis of DHPs by introducing

1synthesis of new DHPs e.g., ZM244085 with K channel ‘‘free’’ amino acid moieties (excitatory or inhibitory) toopening properties and lack of calcium antagonistic or DHP cycle in position 4 instead of the aryl or heterylagonistic mode of the prototypic DHP nifedipine (Li et al., substituent (the characteristic entity of the bi- or polycyclic1996). structures of the typical calcium antagonistic /agonistic

Our novel approach is to consider the DHP molecule not drugs of the DHP series, e.g. nimodipine. It is hypoth-as an indifferent and homogenous structure but as an esised that the choice of ‘‘free’’ amino acid moietyendogenous ligand-resembling structure. Firstly, the DHP attached to the DHP ring modulates the activity of the newcycle itself can be regarded as a dihydronicotinamide DHPs (Misane, 1993).analogue, i.e. a functional group involved in redox re- Glutapyrone, a disodium salt of 2-(2,6-dimethyl-3,5-actions (reversible or irreversible), and free radical diethoxycarbonyl - 1, 4 - dihydropyridine - 4 - carboxamido)-scavenging and peroxidation processes crucial for living glutaric acid, a representative of these series of compoundscell function. In addition, the DHP can be considered as a has been chosen for extended studies because of itsstructure encompassing both, excitatory (glutamate /aspar- structural features (Fig. 1). As its chemical formulatate) or inhibitory (GABA/b-alanine) neuroactive amino indicates, it contains a ‘‘free’’ glutamate moiety attached toacid moieties /elements (Misane, 1993). Secondly, the high the DHP ring in position 4. In addition, an another aminolipophilicity allows the DHP molecule to penetrate easily acid – g-amino-butyric acid (GABA) ‘‘crypto’’ elementsinto the lipid bilayer of both the plasma membrane and the can be seen ‘‘incorporated’’ into the DHP cycle. Both ofmembranes of intracellular organelles. For example, the these amino acids are joined by a peptide -CO–NH- bond.

21DHP derivative ryodipine (foridone) with classical Ca Thus, the glutapyrone molecule resembles a dipeptidechannel-blocking properties was detected in intracellular structure (Duburs et al., 1995; Klusa et al., 1994). Thismembranes, including those of mitochondria (Belevich et feature, probably, explains the unique pharmacologicalal., 1986). Both nimodipine (Hoffmeister et al., 1984) and properties of glutapyrone. Glutapyrone differs from classi-

21nifedipine (Duhm et al., 1972) are able to pass the blood cal DHP compounds by lack of Ca antagonist /agonistbrain barrier (BBB), and the more lipophilic nimodipine action in both neurons (cortical synaptosomes) (Karpova etreaches higher levels in CNS than nifedipine. al., 1993a) and muscles (myocardial slices) (Klusa et al.,

The works of Bodor and co-authors have shown that a 1996). Unlike the classical calcium antagonists /agonists oflipoidal interconvertible dihydropyridine-pyridinium salt DHP series, glutapyrone did not exhibit a significantcan be used – thanks to its ability to cross the blood–brain hypotensive or hypertensive action. At the same time, thebarrier (BBB) – as the carrier molecule for site-specific compound was found to have antiarrhythmic and anti-and sustained delivery of biologically active compounds ishemic as well as antioxidant properties (Utno et al.,(attached to the DHP cycle) to the brain (Bodor and 1989; Veveris and Cirule, 1993). However, preliminary inAbdelAlim, 1985; Bodor and Brewster, 1983; Bodor et al., vitro studies failed to reveal any significant binding affinity1981). Oxidation of the carrier part in vivo to the hydro- of glutapyrone for several neurotransmitter receptors.philic ionic pyridinium salt prevents its elimination fromthe brain, while elimination from the general circulation isaccelerated. Subsequent cleavage of the quaternary carrier-drug species results in sustained delivery of the drug in thebrain and facilitated elimination of the carrier part.

The advantages of this redox delivery system (analogous1to ubiquitous NAD ⇔NADH coenzyme system) approach

based on the carrier properties of the DHP cycle currentlyused in the design of the specific drug-delivery systemsinvolve: good CNS uptake, prolonged ‘‘residence’’ time ofdrug precursor in brain and cerebrospinal fluid, reducedperipheral concentrations and mitigated side effects(Bodor, 1987; Pop and Bodor, 1992). The cerebral toxicitymay also be lowered, since the drug is present most of thetime in the form of an inactive derivative. Both, structural(endogenous ligand-resembling aspects of DHPs) andfunctional (high lipophilicity, carrier function of DHP ringitself) properties suggest that DHP compounds can affectneuronal functioning by altering synaptic function. Since Fig. 1. Chemical structure of glutapyrone.

I. Misane et al. / European Neuropsychopharmacology 8 (1998) 329 –347 331

There was no binding affinity to cholinoreceptors (mus- most extensive research efforts in this field (Ishimaru andcarinic M and M and nicotinic), adrenoreceptors (a , a Toru, 1997; Meldrum, 1995). Unfortunately, most recent1 2 1 2

and b) and GABA /Bzd receptors in rat brain prepara- attempts to affect glutamatergic function in vivo haveA

tions. Most strikingly, glutapyrone did not bind to DHP- resulted into severe problems due to the toxicity of direct-receptor specific sites. Additionally, glutapyrone can be acting NMDA receptor ligands. In view of the extremelydistinguished by its high solubility and extremely low low toxicity of glutapyrone it was of particular interest totoxicity compared with classical DHPs (LD .3 000 examine its possible action on the NMDA-receptor com-50

21 21 21mg kg i.v.; .8 000 mg kg i.p. and .10 000 mg kg plex.p.o. as referred to seven day-observations in mice). In the present study we examined the ability of

In contrast to the lack of effect in vitro, glutapyrone glutapyrone to effect the main ionotropic glutamate re-displayed a number of neuropharmacological effects in ceptor subtypes, i.e. AMPA, kainate (KA), and NMDA

21vivo. The compound (1–5 mg kg ) exerted a marked receptors in the rat brain. Glutamatergic manipulations instress-protective activity in the immobilization stress vivo comprised behavioural studies to investigate thestudies in the rat (Muceniece et al., 1991). Similar to modulation of PCP-induced locomotor hyperactivity byclassical DHPs (Dolin et al., 1988), it also showed glutapyrone. In order to broaden the knowledge on theanticonvulsant properties in the pentylenetetrazol (Karpova psychopharmacological profile of glutapyrone, the com-et al., 1993a), bicuculline and thiosemicarbazide convul- pound was tested in several animal models relevant forsion tests in mice (Karpova et al., 1993b). In addition, affective /depressive disorders in humans. The dose-rangeglutapyrone enhanced the antiepileptic activity of sodium of glutapyrone selected for the present behavioural experi-

21valproate and phenobarbital against pentylenetetrazol- and ments (0.05–5.0 mg kg ) has been shown to be effectiveelectroshock-induced seizures, respectively (Karpova et al., in a number of the previous studies (referred above). Some1993c). In these experiments glutapyrone differed from of neurochemical data presented in the Discussion (in aclassical DHPs by low doses, delayed onset of action view of their particular importance) have been previously(maximum effects following 3–6 h after administration) published in the form of abstract (Muceniece et al., 1991).and, most strikingly, a lack of pronounced dose-effectrelationship. In addition, glutapyrone was found to beeffective against focal penicillin- (Karpova et al., 1993b) 2. Experimental proceduresand aminopyridine-induced epileptic activity and it poten-tiated sodium valproate action against aminopyridine-in- Parts of the present work have been carried out at theduced epileptic activity (Karpova et al., 1993c). However, Latvian Institute of Organic Synthesis, Riga, Latvia;it required much higher (30–80 mg/kg) doses of Department of Neuroscience, Karolinska Institute, Stock-glutapyrone. Therefore, our preliminary data indicated that holm, Sweden and Department of Pharmacology andGABAergic mechanisms are directly or indirectly involved Toxicology, University Kuopio, Finland. The following inin the mediation of glutapyrone effects, although this vivo (behavioural) studies were performed at the Latvianassumption lacked support from quite limited in vitro Institute of Organic Synthesis: forced swimming (Por-binding studies. solt’s) test, reserpine-induced hypothermia and ptosis and

The presence of a glutamate moiety in the molecular open field test. The studies of locomotion in automaticstructure of glutapyrone and the finding that glutapyrone at activity cages were performed at Karolinska Institute.

21a high dose (60 mg kg ) prevented N-methyl-D-aspartate In vitro radioligand binding studies were carried out at(NMDA)-induced tonic convulsions in mice (Klusa et al., University of Kuopio.1994), led to studies on the possible actions of glutapyroneon brain glutamatergic processes. The role of NMDA 2.1. Animals and animal maintaining conditionsreceptor-mediated neurotransmission in the integrativefunctions of CNS, particularly, learning and memory (long- For the parts of the work performed at the Latvianterm potentiation) (Ascher et al., 1991; Fonnum et al., Institute of Organic Synthesis animals (rats and mice) were1995) as well as a wide range of human neuro- obtained from the State Pharmaceutical Company GRIN-psychopathologies, e.g. Alzheimer’s disease and psychosis DEX, Riga, Latvia.and schizophrenia is well-recognised (Danysz et al., 1995; Adult male Icr:Icl mice at 2.5–3 months of age weigh-Fonnum et al., 1995; Ishimaru and Toru, 1997; Javitt and ing 18–22 g at the time of experiment served as subjects inZukin, 1991; Meldrum and Garthwaite, 1990). Although open field, forced swimming (Porsolt’s) and reserpine-there are several novel therapeutical targets, e.g. the induced hypothermia and ptosis tests. Adult male specificglycine site on the NMDA receptor complex or the a- pathogen-free Spraque–Dawley rats (age of 9–10 weeks,amino-3-hydroxy-5-methyl-4-isoxazole propionate weighing 270–320 g at the time of testing) were used in(AMPA) receptor subtype, the modulatory site of the the locomotor behaviour studies performed at KarolinskaNMDA receptor complex sensitive to noncompetitive Institute. The animals were obtained from B & K UNI-antagonists (PCP, MK-801) remains to be the object of VERSAL AB (Sollentuna, Sweden). Adult male Wistar


rats (age of 12–14 weeks, weighing 200–250g) obtained tion at 40 0003g at 258C for 10 min. After washing of thefrom National Animal Centre, Kuopio, Finland were used pellets (20 ml of buffer, 40 000 g for 10 min), the finalfor cortical membrane preparations in radioligand binding pellets were resuspended in 20 ml of Tris–HCl buffer andstudies. the membrane preparations were frozen and stored at

The animals were always maintained at an ambient 2208C for 1–14 days.room temperature of 20618C with 40–50% relativehumidity. A 12 h light /dark schedule was used throughout 2.3.2. Standard binding assaysthe experiment and the animals had free access to standardlab chow and tap water up to the time of experiments.

32.3.2.1. [ H]TCP bindingThe behavioural testing was always performed betweenThe membrane preparation was thawed and centrifuged08.00 and 15.00 h during the light period and an habitua-

at 48 000 ( g) for 10 min and resuspended in 50 mltion period of 1 h to the conditions of the experimentalTris–HCl buffer (5 mM, pH 7.1 determined at 258C) toroom preceded all the experimental procedures.

21give protein concentration 0.8–0.9 mg ml . Standardbinding assays were carried out as described previously.2.2. Test compounds and radioligandsThe incubation period was 30 min at 258C in a totalvolume of 0.25 ml. Nonspecific binding was determined inThe following drugs used in behavioural studies werethe presence of (1)-MK-801 (10 mM). The binding ofdissolved in saline (NaCl 0.9%): glutapyrone [disodium

3 21[ H]TCP (3 nM, 49.6 Ci mM , NEN) was determined insalt of 2-(2,6-dimethyl-3,5-diethoxycarbonyl-1,4-dihydro-26the presence of 6 concentrations of glutapyrone (10 ;pyridine-4-carboxamido)-glutaric acid] (Latvian Institute

26 25 25 24 235310 ; 10 ; 5310 ; 10 ; 10 M). The incubationof Organic Synthesis, Riga, Latvia); D-amphetamine sul-was terminated by filtration through Whatman GF/C glassphate (D-amphetamine) and 1-[1-phenylcyclohex-fibre filters followed by washing with 335 ml ice-coldyl]piperidine (phencyclidine, PCP) both obtained frombuffer. Each filter was placed directly into the scintillationSigma Chemical Co., St. Louis, MO, USA. Reserpinecocktail Ultima Gold (4.5 ml) and counted 24 h later(POLFA, Poland) was dissolved in a drop of glacial aceticspectrometrically.acid and made up to volume with 5.0% glucose.

N-methyl-D-aspartate (NMDA); L-glutamic acid and3kainic acid (KA) were purchased from Sigma Chemical 2.3.2.2. [ H]KA binding

3Co. (St. Louis, MO, USA). (1)-MK-801 was obtained The binding assay procedure for [ H]KA (2 nM, 5821from Research Biochemicals Inc. (Natick, USA). Ci mM , NEN) was the same as described above for

3 3Radioligands ([ H]TCP, 1-[1-(2-thienyl)cyclohex- [ H]TCP except that Tris–citrate buffer (50 mM, pH 7.13yl]piperidine; [ H]AMPA (a-amino-3-hydroxy-5-methyl-4- determined at 258C) was used, incubation period was 1 h

3isoxazole proprionic acid and [ H]KA (kainic acid) were at 08C and nonspecific binding was determined using 100purchased from New England Nuclear (Europe Division). mM of KA.The agents used in binding studies were handled accordingto the requirements of experimental protocol. All other 32.3.2.3. [ H]AMPA bindingchemicals were obtained from standard commercial 3The binding assay for [ H]AMPA (15 nM, 52.1sources. 21Ci mM , NEN) was the same as described above except

that Tris–HCl buffer (30 mM, pH 7.1 determined at 258C),2.3. Radioligand-receptor binding assayscontaining CaCl (2.5 mM) and KSCN (100 mM) in order2

to enhance the specific binding was used. Incubation time2.3.1. Preparations of membraneswas 30 min at 08C and nonspecific binding was defined inThe preparation of membranes from the rat cerebralthe presence of L-glutamate (1 mM).cortex was made according to the method described

elsewhere (Honore et al., 1989). The rats were killed bydecapitation. All further preparations were performed at 2.3.2.4. Data analysis0–48C unless otherwise indicated. The brains were quickly In each experiment, assays were performed in duplicateremoved and cerebral cortices (0.6–0.9 g) were dissected for each of 6 concentrations of glutapyrone. Completeon ice and homogenised for 5–10 s with the Ultra-Turrax experiments were performed at least three times to confirmhomogenizer in 30 ml of Tris–HCl buffer (5 mM, pH 7.1 the data. The inhibition values were expressed as % to thedetermined at 258C). The homogenate was rinsed with 10 corresponding ligand controls. The overall treatment i.e.ml of buffer and the combined suspension centrifuged at glutapyrone concentration effects were analysed by one-40 0003g for 10 min. The pellets were washed three times way analysis of variance (ANOVA). Post hoc analysis waswith 30 ml of the same buffer (3340 0003g310 min). conducted by Student’s t-test. A probability level of P,

The washed pellets were homogenised in 20 ml of buffer 0.05 was accepted as statistically significant and all postand incubated for 30 min at 378C followed by centrifuga- hoc tests were two-tailed.


2.4. Experimental design of behavioural studies was determined 6 h after reserpine administration. Therectal temperature in the animals was measured with aprobe thermometer at a constant depth. Reserpine-induced2.4.1. Effects of Glutapyrone on spontaneous locomotorptosis was scored 0–4 using the modified (opposed)activity in non-habituated animals and PCP- and D-system for eyelid opening described by Rubin et al.amphetamine-induced hyperlocomotion in habituated rats(1957):For the testing of locomotor activity the rats were used

only once, and seven to nine rats were randomly assignedto each treatment group. The rats were acclimatised to the

0: Eyelids completely opened;conditions of the experimental room for 1 h in order to1: Eyelids 3 /4 opened;study the locomotor behaviour without the interference of2: Eyelids 1 /2 opened;stress-factors. Subsequently, the rats were injected with a3: Eyelids 1 /4 opened:selected dose of glutapyrone or saline in the corresponding4: Eyelids completely closed.saline or PCP/ D-amphetamine control groups. They were

thereupon placed individually into the locomotor cages andspontaneous locomotion was recorded for a period of 60 The rate of 4 scores was considered as indication ofmin (0–60 min period). Then, the rats (already habituated completely developed ptosis.to the conditions of locomotor cages) received PCP/ D-amphetamine or saline injection and were again placed into

2.4.2.1. Statistical analysisthe locomotor cages. The monitoring of locomotor activityOverall treatment and time effects of the body tempera-continued for the next 60 min (65–120 min period).

ture and ptosis observations were determined using re-Therefore, the experimental design gave us the possibilitypeated-measures two-way ANOVA. The data was analysedto compile the analysis of locomotor behaviour in bothseparately in the case of glutapyrone immediate and 3 hhabituated and non-habituated animals. Locomotion,post-reserpine administration. For each significant F-ratio,motility and rearing were recorded simultaneously inmultiple group comparisons were performed using Fisher’stwelve computerised locomotor cages (Motron Products,PLSD-test. All results were expressed as means 6 SEM. A¨Sweden) as described previously (Ogren et al., 1979). Theprobability level of P,0.05 was accepted as statisticallybehavioural effects of the treatment were assessed andsignificant, and all post hoc tests were two-tailed.sniffing, ataxia, oral stereotypies and head weaving were

scored.2.4.3. Behavioural despair in forced swimming(Porsolt’s) test in mice2.4.1.1. Statistical analysis

The behaviour of mice in stress situation (forced swim-Overall treatment effects of locomotor behaviour com-ming with no possibility to escape) was evaluated accord-ponents, i.e. locomotion, motility and rearing were ex-ing to the method described in details elsewhere (Porsolt etamined using a repeated-measures two-way ANOVA. Foral., 1977). Briefly, the mice were placed into a cylindereach significant F-ratio, Fisher’s protected least significant(diameter – 10 cm, height – 25 cm) filled with tap waterdifference test (Fisher’s PLSD test) was used to analyse(218C) to 1/3 of its volume and exposed to stress situationthe statistical significance of appropriate pair-wise com-(swimming with no possibility to escape) in one trial. Theparisons. All results were expressed as means 6 SEM. Atotal immobilization time during 6 min and the 15 min testprobability level of P,0.05 was accepted as statisticallyprocedure was registered and served as measurement ofsignificant, and all posthoc tests were two-tailed.behavioural despair in mice. A mouse was judged to beimmobile when it floated in the water in an upright

2.4.2. Effects of glutapyrone on reserpine-inducedposition and made only small movements to keep its head

hypothermia and ptosisabove water. Glutapyrone or saline (for control group) was

The tests were performed according to a model de-injected 1 h prior to the test procedure.

scribed earlier (Costa et al., 1960). Reserpine at a dose of212.5 mg kg or saline was injected into mice. Consecutive-

ly, two different approaches in experimental design were 2.4.3.1. Statistical analysisapplied. Overall treatment effects of behavioural despair were

Glutapyrone or saline (for the concurrent reserpine and determined by two-way ANOVA for both testing pro-drug-free controls) was administered: 1. Immediately after cedures of 6 min and 15 min of duration. For eachreserpine injection. In this case, the development of significant F-ratio, multiple group comparisons were per-reserpine-induced hypothermia and ptosis was estimated 1, formed using Fisher’s PLSD test. The results were ex-2, and 3 h after reserpine administration; pressed as mean 6 SEM. A probability level of P,0.05

2. Three hours after reserpine injection. Under these was accepted as statistically significant, and all post hocconditions the reserpine-induced hypothermia and ptosis tests were two-tailed.


2.4.4. Effects of glutapyrone on locomotor activity of locomotor activity during the entire experimental pro-mice in open field test cedure (0–120 min);

The locomotor activity was estimated in mice either 1, 3 2. The effects of glutapyrone pretreatment on PCP-andor 6 h after glutapyrone (saline for independent corre- D-amphetamine-induced locomotor responses in thesponding control groups at every testing time) injection as animals already habituated to the locomotor cages (65–described earlier (Weischer, 1976). The animals were 120 min).placed into the brightly illuminated black-painted regularoctagonal-shaped (diagonal length of 36 cm) wooden box. As can be seen from Fig. 2, Fig. 3 and Fig. 4, the secondThe black floor was divided into eight equal triangle- injection after 60 min of habituation time resulted in a veryshaped sections. A 60 W bulb was positioned 1 m above short increase (5 min) in locomotor responses in thethe centre of the apparatus. The animals were allowed 1 control groups. All the responses were completely reversedmin of habituation time to the experimental conditions in 10 min after second injection indicating an efficientorder to minimise interference with additional stress acclimatisation to the locomotor cages.factors. Subsequently, horizontal activity (the passages of The experiment on glutapyrone-pretreatment effects onhorizontal lines with all four paws), vertical activity (the PCP-induced locomotor behaviour was subdivided intonumber of two frontal paws lift) and exploratory activity two parts (Exp. 1 and Exp. 2) due to the large number of(hole inspection in the vertical walls) scores were regis- treatment groups and limited time (09.00–15.00 h) suitabletered in a 3 min period of observation. for performance of behavioural studies.

3.2.1. Effects of glutapyrone on spontaneous locomotor2.4.4.1. Statistical analysisactivity in ratsOverall treatment and testing time effects on the counts

The glutapyrone treatment at the at the highest 5.0of horizontal, vertical an exploratory activities in indepen-21mg kg dose as well as at the lower dose range ofdent groups of animals were analysed using polynomial

210.05–0.5 mg kg did not result in significant changes inanalysis of regression and followed by two-way ANOVA.any of three locomotor behaviour parameters tested com-For each significant F-ratio, Fisher’s PLSD-test waspared to saline-treated control groups (Fig. 2 and Fig. 3,applied to examine the statistical significance of multiplerespectively).comparisons between control groups and glutapyrone

In general, the habituation part of the glutapyrone andtreated groups as well as differences of activity betweenD-amphetamine co-interaction study displaying the effectsdifferent testing times of the same treatment in indepen-

21of 5.0 mg kg dose on spontaneous locomotion was in adent groups. The results were expressed as means 6 SEM.good agreement with the corresponding glutapyrone andA probability level of P,0.05 was accepted as a statisti-PCP study. No significant effect of treatment was revealedcally significant, and all post hoc tests were two-tailed.either for locomotion (F 50.70, P.0.55) or motility3,29

(F 52.79, P.0.05) with exception for rearing (F 53,29 3,29

5.88, P,0.01) when a slight and inconsistent (according to3. Results multiple group comparisons) increase in activity counts

appeared in one of two glutapyrone-treated groups. Never-3.1. Effects of Glutapyrone on NMDA-, kainate and theless, at the end of the habituation period no differencesAMPA-receptor binding in the rat cortical membranes between glutapyrone- and saline-treated groups persisted

since rearing counts in all the groups were of zero value.26 23Glutapyrone at the concentration range of 10 –10 M Therefore, the minor elevation of rearing observed in one3 3 3failed to inhibit the [ H]TCP, [ H]AMPA and [ H]kainic of glutapyrone-treated groups was discounted as non-sig-acid binding to the rat cortical membranes (data not nificant for further drug-combination studies. It is notewor-shown). These data indicated that glutapyrone had no thy that Fisher’s PLSD test indicated facilitation of neitherspecific binding affinity to either NMDA- or non-NMDA locomotion, motility nor rearing by glutapyrone compared(AMPA or kainic) ionotropic glutamate receptors. to saline-treated control group at any of 5 min recording

interval during drug co-interaction study (65–120 min).3.2. Behavioural observations in locomotor activitycages 3.2.2. Effects of glutapyrone-pretreatment on PCP-

induced locomotor hyperactivity in ratsAfter adaptation to the conditions of the experimental Injected into habituated rats, PCP at the dose of 2.0

21room, the entire test procedure was designed in the way mg kg induced a significant increase in both locomotionthat allowed to record: and motility, while rearing was unaffected (Fig. 2 and Fig.

3). These results are in agreement with those reported¨previously (Ogren and Goldstein, 1994). In Exp. 1 (the 5.0

211. Glutapyrone dose-response effects on spontaneous mg kg dose of glutapyrone) repeated-measures ANOVA


21Fig. 2. The effects of glutapyrone (5.0 mg kg ) on the spontaneous and PCP-induced locomotor responses (locomotion, motility and rearing) in the rats.21 21Separate groups of rats (n57–8/group) were treated with glutapyrone (5.0 mg kg i.p.) or saline (5 ml kg i.p.) and placed into the locomotor cages. The

effects of glutapyrone on spontaneous locomotor activity were recorded during a period of habituation to the conditions of locomotor cages (0–60 min) as21 21well as the consecutive 65–120 min period. After the habituation was completed, the rats were injected (s.c.) with PCP (2.0 mg kg ) or saline (2 ml kg )

and placed again into the locomotor cages. The effects of glutapyrone pretreatment on the locomotor responses induced by PCP were measured for aconsecutive 60-min period (65–120 min as counted from the beginning of experiment). The results show the mean values 6 SEM of the four treatmentconditions for each consecutive 5 min measurement period. Statistical analysis was performed by repeated-measures ANOVA followed by Fisher’s PLSDtest.


Fig. 2. (continued)

revealed a significant main effect of treatment: F 5 0.01). Glutapyrone pretreatment resulted in a further3,27

15.01, P,0.01 and F 521.27, P,0.01 for locomotion elevation of D-amphetamine-induced hyperactivity. A cer-3,27

and motility observations, respectively. However, no sig- tain specificity in the modulatory profile of glutapyronenificant overall effect of treatment for rearing counts was was seen and it enhanced the D-amphetamine-inducedfound (F 51.27, P.0.30). The same was true for Exp. 2 responses in the following order: rearing..locomotion3,27

when glutapyrone was injected at the lower doses of 0.05 motility.21and 0.5 mg kg . The corresponding F-values calculated Briefly, glutapyrone given at the dose range of 0.05–5.0

21for locomotion, motility and rearing were: F 58.02, mg kg did not exert any lasting and significant modulat-5,42

P,0.01; F 522.23, P,0.01 and F 50.65, P.0.65, ory (stimulatory or inhibitory) influence on spontaneous5,42 5,42

respectively. locomotion in rats during a 120 min-recording time.21 21Fig. 2 shows that at the dose of 5.0 mg kg However, at the 5.0 mg kg dose glutapyrone enhanced

glutapyrone caused a marked increase only in the both, PCP-and D-amphetamine-induced hyperactivity.locomotor responses elevated by PCP, i.e. motility andlocomotion. The potentiation of PCP-induced hypermotili- 3.3. Effects of glutapyrone on reserpine-inducedty was very pronounced while no modulatory influence of hypothermia and ptosis in miceglutapyrone on rearing activity was seen. The multiplegroup comparisons showed that at the lower dose range of The data of glutapyrone effects on reserpine-induced

210.05–0.5 mg kg glutapyrone did not elicit any significant ptosis and hypothermia are presented in Tables 1 and 2,influence on the PCP-induced behavioural responses (Fig. respectively. Since homogeneity of initial values are of3). critical importance for reserpine-task, the temperature was

measured and ptosis observations were carried out prior to3.2.3. Effects of glutapyrone-pretreatment on D- the experiment. The multiple comparison test did notamphetamine-induced locomotor hyperactivity in rats reveal any difference between treatment groups regarding

Fig. 4 shows that D-amphetamine at the dose of 1.5 initial values of body temperature or ptosis. Therefore, a21mg kg (i.p.) after a complete habituation to the contribution of the initial values appeared to be negligible.

locomotor cages induced a marked elevation of all theaspects of locomotor activity recorded: locomotion, motili- 3.3.1. Influence of glutapyrone on hypothermic responsety and rearing. Repeated-measures ANOVA revealed sig- caused by reserpinenificant overall effect of treatment for all the test- Table 1 shows that glutapyrone given at the doses of

21categories: locomotion (F 528.72, P,0.01); motility 0.5 and 5.0 mg kg (i.p.) did not produce changes in body3,29

(F 546.44, P,0.01) and rearing (F 517.34, P, (rectal) temperature in mice up to 3 h after administration.3,29 3,29


21Fig. 3. The effects of glutapyrone low-dose (0.05–0.5 mg kg ) treatment on spontaneous and PCP-induced locomotor responses (locomotion, motility and21 21rearing) in the rats. Separate groups of rats (n58/group) were pretreated with glutapyrone (0.05 and 0.5 mg kg i.p.) or saline (5 ml kg ) 60 min prior to

21 21s.c. injection with PCP (2.0 mg kg ) or saline (2 ml kg ). Effects of glutapyrone on spontaneous locomotion and its effects on the PCP-inducedlocomotor responses were measured for a 0–60 min and consecutive 65–120 min period using the design presented in Fig. 2. The results are expressed asmean values 6 SEM of the six treatment conditions for each consecutive 5 min measurement period. For the details of statistical analysis see Fig. 2.

21Reserpine injection at the 2.5 mg kg dose (i.p.) resulted treatment in both testing paradigms i.e. when glutapyronein gradual time-dependent decrease in rectal temperature was injected immediately after and 3 h after reserpinewith a plateaux effect in the next 3–6 h. The repeated administration (F 562.66, P,0.01 and F 542.03,5,30 5,30

measures ANOVA revealed significant main effect of P,0.01, respectively). Glutapyrone, administered immedi-


Fig. 3. (continued)

ately after reserpine only partly blocked the development pine-hypothermia test, glutapyrone at both doses tested21of reserpine-hypothermia since Fisher’s PLSD test re- (0.5 and 5.0 mg kg ) partly antagonized already de-

vealed significant differences between glutapyrone1 veloped reserpine-ptosis (P,0.01 versus saline1saline andreserpine treated groups versus reserpine1saline and P,0.05 versus reserpine1saline groups).saline1saline (control) groups examined in the following1–3 h. 3.4. Effects of glutapyrone on behavioural despair in

When injected 3 h after reserpine, glutapyrone at the 5.0 forced swimming test in mice21mg kg dose rather enhanced (P,0.05 versus reserpine1

saline group) than blocked the reserpine-induced hypo- The mice were exposed to the stress-situation of forcedthermia according to the observations performed 6 h after swimming with no possibility to escape. The total im-reserpine injection. mobilization time served as measurement of behavioural

despair and was assessed in the tests of 6 and 15 min of3.3.2. Effects of glutapyrone on reserpine-induced ptosis duration.

The data displayed in Table 2 show that in two Glutapyrone given in a single injection 1 h prior to theindependent parts of the study glutapyrone at the doses of test-procedure resulted in dose-dependent decrease in total

210.5–5.0 mg kg did not elicit any pro-ptosis activity immobilization time. The two-factor ANOVA revealedduring 3 h after administration. The time-course of re- significant effect of treatment in both, 6 min-test (F 52,15

serpine-induced ptosis corresponded to the development of 6.48, P,0.01) and 15 min-test (F 55.58, P,0.05). The2,15

hypothermic response (plateaux effect reached 3–6 h after multiple comparisons using Fisher’s PLSD test displayed21injection). that in the 6 min-test a dose of 5.0 mg kg was required

The overall analysis of the ptosis ‘‘response’’ using for shortening of immobilization time (P,0.01 vs salinerepeated measures ANOVA revealed a significant effect of group), while in the 15 min-test even the dose of 0.5

21treatment in both testing paradigms: F 526.60, P,0.01 mg kg appeared to be active (P,0.05 vs saline group).5,30

in the case of immediate post-reserpine administration of However, the effect tended to be more pronounced at the21glutapyrone and F 514.91, P,0.01 in the case of 3 h 5.0 mg kg dose (P,0.01 vs saline) (Fig. 5).5,30

post-reserpine administration of glutapyrone.21Glutapyrone treatment at the 5.0 mg kg dose immedi- 3.5. Modulatory effects of glutapyrone on locomotor

ately after reserpine injection resulted in partial inhibition activity of mice in ‘‘open field’’ testof the ptotic reaction (P,0.01 versus saline1saline- andreserpine1saline-treated groups). Contrary to the reser- The approach of single exposure to the conditions in the


21Fig. 4. The effects of glutapyrone (5.0 mg kg ) on the spontaneous and d-amphetamine-induced locomotor responses (locomotion, motility and rearing) in21 21the rats. Separate groups of rats (n58–9/group) were pretreated with glutapyrone (5.0 mg kg i.p.) or saline (5 ml kg i.p.) 60 min prior to i.p. injection

21 21with D-amphetamine (1.5 mg kg ) or saline (5 ml kg ). Effects of glutapyrone on spontaneous locomotion and its pretreatment effects on theD-amphetamine-induced locomotor responses were recorded for a 0–60 min and consecutive 65–120 min period using the design presented in Fig. 2. Theresults are expressed as mean values 6 SEM of the four treatment conditions for each consecutive 5 min measurement period. For the details of statisticalanalysis see Fig. 2.

21‘‘open field’’ box was applied to study the spontaneous and glutapyrone-treated (the dose range of 0.05–5.0 mg kg )natural reactions of mice to novel environmental con- groups were used only once at each of 3 test-timesditions. Independent control (saline-treated) and selected: either 1, 3 or 6 h after injection, respectively. A


Fig. 4. (continued)

summary of the influence of glutapyrone treatment on the main effect of treatment for horizontal activity (F 52,69

counts of horizontal, exploratory and vertical activities is 12.52, P,0.01) and exploratory activity (F 53.98, P,2,69

displayed in Fig. 6. 0.05) and non-significant for vertical activity (F 50.31,2,69

Polynomial regression analysis displayed significant P.0.73). The same time, the overall effect of time was

Table 1The effects of glutapyrone on reserpine-induced hypothermia in mice

Rectal temperature (8C)Time of measurements

21Treatment Before experiment After reserpine (2.5 mg kg ) administration

1 h 2 h 3 h 6 h

A. Glutapyrone administered immediately after reserpine injectionI. SAL1SAL 39.4360.24 38.8060.38 39.5760.23 39.3360.08II. SAL1GLU 0.5 39.4060.23 38.7060.17 38.7360.29 39.1060.30III. SAL1GLU 5.0 39.4760.16 38.7060.18 39.2760.25 39.0360.17

a a aIV. RES1SAL 39.4360.24 35.1260.36 34.6360.20 32.8060.26a a a [V. RES1GLU 0.5 39.4060.23 35.9760.27 36.1060.35 34.7760.63a [ a a [[VI. RES1GLU 5.0 39.4760.16 36.2060.50 35.3360.48 33.8760.46

Repeated-measures ANOVA: F 562.66, P,0.01(5,30)

B. Glutapyrone administered 3 h after reserpine injectionI. SAL1SAL 39.4360.24 38.1760.23II. SAL1GLU 0.5 39.4060.23 38.0760.24III. SAL1GLU 5.0 39.4760.16 38.1760.31

aIV. RES1SAL 39.4360.24 32.6760.41aV. RES1GLU 0.5 39.4060.23 33.2060.45a [VI. RES1GLU 5.0 39.4760.16 31.0760.71


The results are expressed as mean values 6 SEM. The overall F-ratios of treatment effect obtained by repeated-measures ANOVA and corresponding Pvalues are presented.a [ [[P,0.01 versus SAL1SAL control and P,0.05 versus RES1SAL control; P,0.01 versus RES1SAL control (Fisher’s PLSD test, n56). SAL,

21 21Saline (25 ml kg i.p.); GLU 0.5 and GLU 5.0, glutapyrone at the doses of 0.5 and 5.0 mg kg (i.p.), respectively; RES, reserpine at the dose of 2.521mg kg (i.p.).


Table 2The effects of glutapyrone on reserpine-induced ptosis in mice

Scores of ptosisTime of observations

21Treatment Before experiment After reserpine (2.5 mg kg ) administration

1 h 2 h 3 h 6 h

A. Glutapyrone administered immediately after reserpine injectionI. SAL1SAL 0.0060.00 0.6760.67 0.5060.22 0.3360.21II. SAL1GLU 0.5 0.0060.00 0.3360.21 0.1760.17 0.6760.50III. SAL1GLU 5.0 0.0060.00 0.0060.00 0.1760.17 0.6760.33

a aIV. RES1SAL 0.0060.00 0.6760.33 3.3360.33 3.8360.17a aV. RES1GLU 0.5 0.0060.00 0.6760.21 2.8360.48 3.5060.22a [ a [[VI. RES1GLU 5.0 0.0060.00 1.0060.52 2.1760.48 2.1760.75


B. Glutapyrone administered 3 h after reserpine injectionI. SAL1SAL 0.0060.00 0.5060.34II. SAL1GLU 0.5 0.0060.00 0.1760.17III. SAL1GLU 5.0 0.0060.00 0.3360.21

aIV. RES1SAL 0.0060.00 3.8360.17a [V. RES1GLU 0.5 0.0060.00 2.6760.62a [VI. RES1GLU 5.0 0.0060.00 2.6760.62


The results are expressed as mean values 6 SEM. The overall F-ratios of treatment effect obtained by repeated-measures ANOVA and corresponding Pvalues are presented.a [ [[P,0.01 versus SAL1SAL control and P,0.05 versus RES1SAL control; P,0.01 versus RES1SAL control (Fisher’s PLSD test, n56). SAL,

21 21Saline (25 ml kg i.p.); GLU 0.5 and GLU 5.0, glutapyrone at the doses of 0.5 and 5.0 mg kg (i.p.), respectively; RES, reserpine at the dose of 2.521mg kg (i.p.).

found to be significant for all the parameters of locomotor groups showed significant effect of time: F 5141.20,2,15

behaviour tested: horizontal activity (F 515.51, P, P,0.01 for horizontal activity; F 517.01, P,0.01 for2,69 2,15

0.01); exploratory activity (F 521.27, P,0.01) and exploratory activity and F 512.75, P,0.01 for vertical2,69 2,15

vertical activity (F 510.51, P,0.01). It is clear from activity. The only rational explanation of this general2,69

Fig. 6 that independent saline-treated control groups tested decrease in activity is a physiological response to a long-only once, displayed gradual (1h.3h.6h) decrease in all term progressive habituation to the environmental con-the activity parameters tested. The subsequent factorial ditions of experimental room. Therefore, both the overallanalysis of locomotor responses in saline-treated control treatment effect and the time factor had to be considered in

the analysis of data.It is clear that glutapyrone did not stimulate (exploratory

activity) but rather slightly reduced locomotor activity(horizontal and vertical: F 53.10, P50.05 and F 53,20 3,20

6.41, P,0.01, respectively) in the groups of animals tested1 h after injection.

Therefore, the effects of glutapyrone displayed in Fig. 6can be viewed from two main angles: (1) when thetreatment effect was considered, dose-dependent long-termgradual (3–6 h after administration) elevation of locomotor

21activity was detected, particularly, at the 5.0 mg kg doseand in the groups tested 6 h after injection when horizontaland exploratory activities were increased at all the doses

21tested (0.05–5.0 mg kg ) and vertical activity was sig-21nificantly (P,0.05) higher at the 5.0 mg kg dose; (2)

when factorial analysis was applied for time effect,Fig. 5. The effects of glutapyrone on the immobility of mice in the forcedswimming test. The mice (n56/group) were injected i.p. with glutapyrone treatment appeared to result in a dose-depen-

21 21glutapyrone at the doses of 0.5 and 5.0 mg kg or saline 25 ml kg 1 h dent ‘‘stabilisation’’ of locomotor responses. For example,21prior to test procedure. The results show the mean values 6 SEM of the ANOVA revealed that at the most active, 5.0 mg kg dose

three treatment conditions for a single trial of 6 min and 15 min-tests.main effect of time was significant only for horizontalStatistical analysis was performed by two-way ANOVA followed byactivity (F 58.90, P,0.01), while it was non-significantFisher’s PLSD test: *P,0.05 versus saline control group, **P,0.01 2,15

versus saline control group. for exploratory and vertical activities (F 50.14, P.0.872,15


and F 50.02, P.0.98, respectively). Further analysis by2,15

Fisher’s PLSD test showed that compared to the corre-sponding counts of horizontal activity at the test-time of 1h, the increase in activity appeared to be significant only at3 h, while no difference was found at the test-time of 6 h.

Therefore, a combined approach to the analysis of dataincluding both overall treatment and time effects did notallow to deduce consistent motor stimulant effects ofglutapyrone in open field test. The only exception was the

21increase in horizontal activity at the 5.0 mg kg dose inthe group tested 3 h after injection when significance oftreatment and time effect coincided.

4. Discussion

The present study has significantly broadened ourknowledge on the spectrum of the psychopharmacologicalproperties of glutapyrone, a representative of the novelamino acid-containing and dipeptide-mimicking ‘‘class’’ of1,4-dihydropyridine (DHP) derivatives. As a result, po-tential mechanisms of its action have been revealed.

3It is notable that glutapyrone failed to alter [ H]TCP,3 3[ H]AMPA and [ H]KA binding in the rat cortical mem-

branes. Hence, any direct effects of glutapyrone on themain glutamate ion/channel-operated receptor complexes(NMDA and non-NMDA) appear not to be likely.

In order to examine any possible interaction ofglutapyrone with glutamate-receptor function in vivo,effects of glutapyrone were tested in PCP-treated rats. Ourresults confirmed the previous findings showing thatsystemic injection at the comparatively low dose of PCP (2

21mg kg s.c.) resulted in a significant stimulation oflocomotor activity (locomotion and motility) in habituated

¨animals (Ogren and Goldstein, 1994). Due to the completehabituation which preceded the administration of PCP, itwas not possible to detect any further suppression ofrearing.

Pretreatment with the DHP derivative glutapyrone at the21dose of 5 mg kg resulted in a profound enhancement of

PCP-induced motor activation. The finding is remarkablefor several reasons:

(1) During the total time of experiment (2 h afterinjection into non-habituated animals) glutapyrone itselfdid not exert any significant motor activity modulatoryproperties. It distinguishes glutapyrone from classical DHP

21Ca antagonists like nimodipine and nifedipine which atthe dose range similar to that of glutapyrone are known to

Fig. 6. The effects of glutapyrone on locomotor activities: horizontal, 21produce sedation in rodents while Bay K 8644 (Caexploratory and vertical activities of mice in the open field test. Theagonist) has been reported to increase locomotor responsesindependent groups of mice (n56/group) were injected i.p. with

21glutapyrone at the doses of 0.05; 0.5 and 5.0 mg kg or saline (25 (Bolger et al., 1985; Czyrak et al., 1990; Pucilowski, 1992;21ml kg ). The locomotor behaviour was examined either 1, 3 or 6 h later Viveros et al., 1996);

in in a single trial of 4 min total duration in the ‘‘open field’’ box. The (2) The facilitation of the increase in locomotor activityactivity counts were registered and are presented for the last 3 min. The 21by PCP observed at the dose of glutapyrone 5.0 mg kg isoverall treatment and time effects were examined by analysis of polyno-

unusually powerful. This observation, again, is completelymial regression and completed by two-way ANOVA analysis. For each21

significant F-ratio, Fisher’s PLSD test was applied. opposite to those reported for classical DHP Ca antago-


nists known to block hyperlocomotion induced by non- duced hyperactivity (Grebb, 1986). Moreover, nifedipinecompetitive NMDA antagonists MK-801 and PCP (Bolger and nicardipine have been reported to reduce DA transmis-et al., 1986; Grebb et al., 1985; O’Neill and Bolger, 1989; sion in vitro and in vivo and these effects were strongly

21Popoli et al., 1990); related to their Ca antagonist properties (Mena et al.,(3) Glutapyrone pretreatment potentiated only the pa- 1995). Taken together, our data clearly indicate that

rameters of motor activity (locomotion and motility) glutapyrone possesses catecholaminergic, i.e. dopa-already elevated by PCP. The most dramatic was an minergic neurotransmission-enhancing properties. Appar-increase in motility, indicating an enhancement of ently, the modulation of NMDA-receptor responses bystereotypic responses; glutapyrone is rather consequential than direct. The exact

(4) Thus, for the first time, we showed that a dipeptide mechanisms involved in the mediation of these effects are(or dipeptide-like structure) given systemically can in- to be defined in the future. However, a careful analysis offluence a behavioural response believed to be induced, in in vitro and in vivo data have led us to some conclusions.

3part, by NMDA receptor blockade. With respect to the The PCP analogue [ H]1-[1-(2-thienyl)cyclohex-3results of receptor binding studies, this effect could not be yl]piperidine (TCP) and [ H]MK-801 have been extensive-

attributed directly to the action of glutapyrone on PCP ly used to label the PCP binding site associated with3binding site associated with the NMDA receptor / ion NMDA receptors (PCP site 1). Besides that, [ H]TCP also

channel. binds to a site that is insensitive to MK-801 (PCP site 2)3Although PCP affects several neurotransmitter systems, and [ H]TCP in the presence of MK-801 can be used to

the behavioural and psychomimetic effects of PCP have label this site. Recently, it was found that PCP site 2 is notbeen explained, mainly, as a result of NMDA receptor associated with the NMDA receptor / ionophore complexblockade (Carlsson and Carlsson, 1990; Javitt and Zukin, but with biogenic amine transporters (BATs) and is MK-1991; Johnson and Jones, 1990) and/or indirect facilitation 801-insensitive (Goodman et al., 1994; Rothman, 1994;of dopamine (DA) transmission (Javitt and Zukin, 1991; Rothman et al., 1989). Importantly, most of mammalian

21Johnson, 1987). Apparently, at the dose of 2 mg kg , any species with the exception of the rat (the species used inaction on sigma, or serotonin receptor (seen when PCP the present study) have detectable levels of PCP site 2.

21injected at the dose .5 mg kg ) functions could be Although, PCP clearly inhibits the reuptake of biogenicdiscounted as non-significant (Hiramatsu et al., 1989; amines, particularly, DA in the rat. The reason for thisJavitt and Zukin, 1991). controversy remains to be solved. However, the current

Since several studies have demonstrated an importance inability to detect PCP site 2 in vitro does not mean that itof dopaminergic component in the hyperlocomotor re- may not exist and it may not fulfil a function in vivo

¨sponse to PCP treatment (Jackson et al., 1994; Ogren and (Rothman, 1994).Goldstein, 1994), the role of DA transmission in the Therefore, in the present study only the PCP site 1mediation of in vivo effects of glutapyrone was consid- associated with the NMDA-receptor complex was labelled

3ered. Consistent with a DA involvement was the observa- by [ H]TCP and the PCP site 2 remained to be ‘‘un-21tion that the glutapyrone dose of 5.0 mg kg which covered’’. Although direct evidence remains to be pro-

reinforced PCP-induced hyperactivity, glutapyrone also vided it is possible that glutapyrone acts via a BAT-relatedcaused a marked potentiation of D-amphetamine-induced PCP site 2 and/or currently undefined specific bindinglocomotor activity (locomotion, motility and rearing). The site(s) distinct from the classical BATs. In fact, the abilityincrease was particularly evident with regard to a measure of glutapyrone to potentiate locomotor increases by bothof exploration, e.g. rearing activity. This is of particular PCP and D-amphetamine to a great extent attributed to DAimportance since increase in rearing is a typical response uptake inhibition supports this assumption.to indirectly acting DA agonists such as D-amphetamine In the second part of our work devoted to the furtherand distinguishes them from non-competitive NMDA-re- investigation of pharmacological profile of glutapyrone inceptor agonists like PCP and MK-801 which have been several animal models relevant for human depression andshown to reduce rearing counts (Jackson et al., 1994; anxiety, the compound was found to exert both slight orOgren and Goldstein, 1994). D-Amphetamine has no direct profound effects, depending on the task.effects on glutamate receptors, however, it is catechol- Reserpine treatment has been described to induce epi-amine-related drug that induces release of DA and to a less sodes of depression in humans (Jensen, 1959). In rodentsextent, noradrenaline in the CNS and periphery (Seiden reserpine induces a ‘‘depression-like’’ syndrome expressedand Sabol, 1993). Therefore, the increase in rearing might as sedation and motor retardation as well as hypothermiabe considered as mediated specifically by catechola- and ptosis (Costa et al., 1960). The ability of drugs tominergic and, particularly, dopaminergic systems. antagonize the effects of reserpine has served as indication

It is noteworthy that the ability of glutapyrone to of their antidepressant activity (for further references seefacilitate D-amphetamine-induced behaviours was again Bourin, 1990). The studies in reserpine-test revealed aunlike DHP compounds such as nimodipine and nifedipine slight protective while not antagonistic influence ofsince they are known to antagonize D-amphetamine-in- glutapyrone, since reversal of both ptosis and hypothermia


was only partial. Moreover, when glutapyrone treatment fore, one of the possible explanations for positive effects ofwas initiated in already depleted monoamine stores, i.e. 3 h the compound in Porsolt’s test could be its GABA-after reserpine injection, it not only failed to block modulatory profile discovered in our earlier studies andreserpine but, rather the opposite, at the dose of 5.0 described in the Introduction (Karpova et al., 1993a,b,c)

21mg kg it produced a further aggravation of the hypo- since GABA-mimetics with different pharmacological andthermic response. therapeutical implications, i.e. sodium valproate,

Antagonism of reserpine-induced hypothermia and piracetam, GABA, muscimol and baclofen have beenptosis involves different mechanisms. Reserpine-hypother- reported to possess a profound activity in the forcedmia test is indicative for substances (mostly, antidepres- swimming task at their lower dose range (Aley andsants) having direct or indirect b-mimetic activity since Kulkarni, 1989). Furthermore, augmentation of GABA-reserpine-induced decrease in body temperature has been ergic deficit induced by stressful events has been consid-attributed to lack of norepinephrine stimulation of b- ered as one of effective ways for alleviating of depression.adrenergic receptors. However, drugs other than antide- However, the lack of evidence from in vitro bindingpressants (D-amphetamine, aspirin) can also antagonize or studies as well as the unusually broad spectrum of in vivoprevent reserpine-induced hypothermia. The ptosis an- effects of glutapyrone rules out the possibility that thetagonism in the reserpine-test is known to be an indication compound could exert its actions only on GABA-ergicfor drugs with (a-adrenergic activity (thus not antidepres- sites. Therefore, it is possible that the pharmacologicalsants) or serotoninergic activity (possibly, antidepressants). effects of glutapyrone might be expressed, in part, asOn the other hand, drugs which can antagonise ptosis concomitant modulation of several, e.g. DA- and GABA-while also acting on hypothermia and/or akinesia have a as well as glutamatergic mechanisms. Although, predomi-direct noradrenergic and/or dopaminergic effect (for fur- nance of one or another component of neurotransmissionther references see Bourin, 1990). Therefore, the present under certain pathological conditions and/or when thedata indicate that direct action of glutapyrone on mono- system has been specifically activated seems to be likely.aminergic, predominantly, adrenergic (a and b), A direct line of evidence which proves the assumptionserotoninergic and to less extent, dopaminergic sites can be of multi-regulatory profile of glutapyrone under pathologi-ruled out and considered rather as secondary to other cal conditions came from immobilization stress studiesmechanisms. (Muceniece et al., 1991) (published previously in a form

The ability of drugs to reduce immobilization time of of abstract). The rats were exposed to a long-term (3 h)mice and rats in the forced swimming test has been used severe stress, i.e. totally immobilized animals were im-for screening of potential antidepressants (Porsolt et al., mersed into the water. Administration of glutapyrone at the

211978, 1977). Although some psychostimulants or anticho- dose of 1.5 mg kg just prior to stress-procedure com-linergics have been reported to reduce immobilization time pletely restored GABA as well as NA levels markedlyin Porsolt’s test they can be distinguished from true decreased by the stress-procedure in hypothalamus andantidepressants since those ‘‘false positives’’ (mostly dopa- striatum, respectively. When given 3 h prior exposure tominergic drugs such as D-amphetamine) nonspecifically stress, glutapyrone normalized NA level in hypothalamusincrease locomotor activity (Borsini and Meli, 1988; and GABA level in striatum. Moreover, a marked increasePorsolt et al., 1979; Willner, 1984). Glutapyrone was in GABA level caused by glutapyrone pretreatment wasfound to produce a significant dose-dependent decrease in found in the hypothalamus. Interestingly, glutapyroneimmobility in forced swimming test. The effect of failed to change 5-HT and 5-HIAA levels which were onlyglutapyrone was more pronounced at 15 min-test than 6 slightly decreased as compared to GABA and NA. In thismin-test, thus, indicating not simply antidepressant activity study, glutapyrone did not change corticosterone concen-of the DHP compound but, possibly, stress-protective tration in blood and NA content in the adrenal gland, thus,action in the situation when the exposure to stressor was exhibiting CNS-specific action. Taking together,prolonged. It should be emphasized that the test procedure glutapyrone appeared to be effective not only in reestab-was performed 1 h after glutapyrone administration. There- lishment of both excitatory and inhibitory (NA and GABA,fore, reduction in immobility caused by glutapyrone barely respectively) neurotransmitters but, obviously, was able tocould be considered as a ‘‘false positive’’ since the induce long-lasting stimulation of brain functions viainvestigations in locomotor activity cages did not show any activation of neurotransmitter metabolism and/or neuro-increases in locomotor responses (locomotion, rearing and transmitter synthesis. The concurrent normalization /eleva-motility) by the compound itself. Interestingly, the short- tion of excitatory and inhibitory neurotransmitters and theening of immobilization time by glutapyrone was seen late onset of action as well as lack of distinct dose-

21already at the dose of 0.5 mg kg in the 15 min-test. As response effects and rapid reaching of plateau effects seenshown above, this dose lacked activity in modulation of in a number of our studies indicated the ability of thePCP-induced motor stimulation. The present data have dipeptide-like molecule of glutapyrone to balance brainindicated that a direct action of glutapyrone on most of neurochemical mechanisms in the manner that is typicalbiogenic amine-related events seems to be unlike. There- for endogenous ligands.


In ‘‘open field’’ studies glutapyrone pretreatment re- Karolinska Institute International Research and Trainingsulted in profound and long-lasting (at least up to 6 h) Programme (KIRT; Reg, no: Latvia 10 (1995) and Reg,modulation of locomotor responses in mice. At the first no: Latvia 13 (1996)); Nordic Council of Ministers, thelook, increases in behavioural responses by glutapyrone as Centre for International Mobility (CIMO) and Latviancompared to the activity measured in corresponding con- Science Council, grant No. 96.0698. The authors gratefullytrol groups could account for its ‘‘motor stimulant’’ acknowledge the assistance of Assoc. Prof. Leenaproperties. However, serious objections can be raised Tuomisto (Department of Pharmacology and Toxicology,against this conclusion since open field task is used not University of Kuopio, Finland).only for testing of locomotor activity itself but rather as anethological animal model of anxiety (Griebel, 1995;Sanger, 1991). The unconditioned reaction to previously Referencesunknown conditions, most likely, reflects the combinedemotional and motor response of animals. From this point

Aley, K.O., Kulkarni, S.K., 1989. GABA-mediated modification ofof view, the effects of glutapyrone, particularly, with despair behavior in mice. Naunyn-Schmiedeberg’s Arch. Pharmacol.respect to exploratory activity, could be viewed as ‘‘an- 339, 306–311.

Ascher, P., Choi, D.W., Christen, Y., 1991. Glutamate: Cell Death andxiolytic-like’’ since it did not induce any further increaseMemory. Springer-Verlag, Berlin Heidelberg.in locomotion as compared to activity level observed 1 h

Belevich, G.V., Dubur, G.Y., Spirin, M.M., Dobretsov, G.E., 1986.after injection (not different or rather slightly lower thanLocalization of calcium antagonist Ryodipine in the cell membranes.

that of control animals). A study by fluorescence methods. Biologicheskye Membrani 3, 368–In summary, introduction of ‘‘free’’ amino acid moiety 375 (in Russian).

Bodor, N., 1987. Redox drug delivery systems for targeting drugs to theto the DHP structure has resulted into the series ofbrain. Ann. N.Y. Acad. Sci. 507, 289–306.dipeptide-resembling compounds with unique pharmaco-

Bodor, N., AbdelAlim, A.M., 1985. Improved delivery through biologicallogical properties. The results of this study indicate thatmembranes. XIX Novel redox carriers for brain-specific chemical

glutapyrone acts via mechanisms which cannot be attribu- delivery systems. J. Pharmacol. Sci. 74, 241–245.ted to the direct modulation of one neurotransmitter system Bodor, N., Brewster, M.E., 1983. Problems with delivery of drugs to the

brain. Pharmacol. Ther. 19, 337–386.or particular receptor subtype. The present data support ourBodor, N., Farag, H.H., Brewster, M.E., 1981. Site-specific, sustainedearlier findings (Muceniece et al., 1991) that the compound

release of drugs to the brain. Science 214, 1370–1372.can concurrently influence both, excitatory and inhibitoryBolger, G.T., Weissman, B.A., Skolnick, P., 1985. The behavioural effects

components of neurotransmission. However, the exact of the calcium agonist BAY K 8644 in the mouse: Antagonism by themechanisms underlying its long-term stress-protective, calcium channel antagonist nifedipine. Naunyn-Schmiedeberg’s Arch.anticonvulsant and neurodeficit-preventive (studies in al- Pharmacol. 328, 373–377.

Bolger, T., Rafferty, M.F., Crawley, J.N., Paul, S.M., Skolnick, P., 1986.coholized maternal offspring) (Klusa and Germane, 1996)Effects of calcium antagonists on phencyclidine behaviors. Pharmacol.effects and its delayed onset of action are still to beBiochem. Behav. 25, 45–49.

defined. Due to the high lipophilicity and eventual carrier Borsini, F., Meli, A., 1988. Is the forced swimming test a suitable modelfunction of DHP structure (discussed in the Introduction), for revealing antidepressant activity? Psychopharmacology 94, 147–the predominant actions of glutapyrone on intracellular 160.

Bourin, M., 1990. Is it possible to predict the activity of a newevents have been considered. Thus, glutapyrone can stimu-antidepressant in animals with simple psychopharmacological tests?late the phorbol-activated protein kinase C (PKC) in aorticFund. Clin. Pharmacol. 4, 49–64.

smooth cells (A7r5 line) indicating that the intensification Braunwald, E., 1982. Mechanism of action of calcium channel blockingof intracellular events might be G protein-coupled (Klusa agents. N. Engl. J. Med. 307, 1618–1627.et al., 1996). Similar mechanisms responsible for a highly Carlsson, M., Carlsson, A., 1990. Interactions between glutamatergic and

monoaminergic systems within the basal ganglia. Implications forselective and long-term manifestation of pharmacologicalschizophrenia and Parkinson’s disease. Trends Neurosci. 13, 272–276.effects of glutapyrone might be involved in the brain.

Cavero, I., Spedding, M., 1983. Calcium antagonists: a class of drugsTaken together, glutapyrone with its unique CNS-reg- with a bright future. Part I. Cellular calcium homeostasis and calcium

ulatory pattern and extremely low toxicity, undoubtedly, as a coupling messenger. Life Sci. 33, 2571–2581.deserves further investigation. Studies are in progress to Costa, E., Garattini, S., Valzelli, L., 1960. Interactions between reserpine,

chlorpromazine and imipramine. Experientia 16, 461–463.analyse whether the compound can in a selective mannerCzuczwar, S.J., Chodkowska, A., Kleinrok, Z., Malek, U., Jagiello-effect G protein-coupled mechanisms in vivo and/or

Wojtowicz, E., 1990. Effects of calcium channel inhibitors upon thewhether it has a unique action on brain membrane binding efficacy of common antiepileptic drugs. Eur. J. Pharmacol. 176,sites (BAT-associated or non-associated). 75–83.

Czuczwar, S.J., Malek, U., Kleinrok, Z., 1990. Influence of calciumchannel inhibitors upon the anticonvulsant efficacy of commonantiepileptics against pentylenetetrazol-induced convulsions in mice.AcknowledgementsNeuropharmacology 29, 943–948.

Czyrak, A., Mogilnicka, E., Siwanowicz, J., Maj, J., 1990. SomeThis work was supported in part by grants from the behavioural effects of repeated administration of calcium channel

Royal Swedish Academy of Sciences, Project N 1375; The antagonists. Pharmacol. Biochem. Behav. 35, 557–560.


Danysz, W., Parsons, C., Bresink, I., Quack, G., 1995. Glutamate in CNS bicuculline- or thiosemicarbazide-induced convulsions. Bull. Exp.Biol. Med. 116, 1207–1210.disorders. Drug News Prospect. 8, 261–277.

Karpova, M.N., Pankov, O.Y., Germane, S.K., Klusha, V.E., Dubur, G.Y.,Dolin, S.J., Hunter, A.B., Halsey, M.J., Little, H.J., 1988. Anticonvulsant1993. Antiepileptic effects of glutapyrone, a novel derivative ofprofile of the dihydropyridine calcium channel antagonists, nitren-1,4-dihydropyridine, administered in combination with sodium val-dipine and nimodipine. Eur. J. Pharmacol. 152, 19–27.proate and phenobarbital. Bull. Exp. Biol. Med. 116, 1482–1485.Duburs, G., Klusa, V., Bisenieks, E., Liepina, I., Germane, S., Biseniece,

Klusa, V., Germane, S., 1996. Alcoholized maternal rat offspring: modelD., Poikans, J., 1995. Novel peptidomimetics: focus onfor testing of physical and psychoemotional neurodeficit. Scand. J.dihydroisonicotinoyl structures containing amino acid moieties (Ab-Lab. Anim. Sci. 23, 403–409.stract). AFMC International Medical Chemistry Symposium, Tokyo,

Klusa, V., Germane, S., Mishlyakova, N., Muceniece, R., Dambrova, M.,Japan, p. 229.Svirskis, S., 1994. Novel dipeptide-mimicking substances: peculiarDuhm, B., Maul, W., Medenwald, H., Patzschke, K., Wegner, L.A., 1972.modulators of the CNS functioning (Abstract). Can. J. Physiol.Tierexperimentelle untersuchungen zur pharmakokinetik und biotrans-Pharmacol. 72, 418.formation von radioaktiv markiertem 4-(29-Nitrophenyl)-2,6-dimethyl-

Klusa, V., Vitolina, R., Dambrova, M., Atare, Z., Mezapuke, R., Meirena,¨1,4-dihydropyridin-3,5-dicarbonsaure dimethylester. Arzn. Forsch.D., Bisenieks, E., Duburs, G., Azzi, A., 1996. The effects ofDrug Res. 22, 42–52.glutapyrone, a glutamate-containing 1,4-dihydropyridine compound,˚Fonnum, F., Myhrer, T., Paulsen, R.E., Wangen, K., Oksengard, A.R.,on the cardiovascular system, muscle contraction and protein kinase C

1995. Role of glutamate and glutamate receptors in memory functionactivation. Proc. Latv. Acad. Sci. 50 (Section B), 108–113.

and Alzheimer’s disease. Ann. N.Y. Acad. Sci. 757, 475–486.Li, J.H., Yasay, G.D., Kau, S.T., Ohnmacht, C.J., Trainor, A.D., Bonev,

Goodman, C.B., Thomas, D.N., Pert, A., Emilien, B., Cadet, J.L., Carroll, A.D., Heppner, T.J., Nelson, M.T., 1996. Studies of the K channelATPF.I., Blough, B.E., Mascarella, S.W., Rogawski, M.A., Subramaniam, opening activity of the new dihydropyridine compound 9-(3-S., Rothman, R.B., 1994. RTI-4793-14, a new ligand with high affinity cyanophenyl)-3,4,6,7,9,10-hexahydro-1,8-(2H,5H )-acridinedione in3and selectivity for the (1)-MK801-insensitive [ H]1-(1-(2- bladder detrusor in vitro. Arzn. Forsch. Drug Res. 46, 525–530.thienyl)cyclohexyl)piperidine binding (PCP site 2) of guinea pig brain. Martin, M.I., Del Val, V.L., Colado, M.I., Goicoechea, C., Alfaro, M.J.,Synapse 16, 59–65. 1996. Behavioural and analgesic effects induced by administration of

Grebb, J.A., 1986. Nifedipine and flunarizine block amphetamine-induced nifedipine and nimodipine. Pharmacol. Biochem. Behav. 55, 93–98.behavioural stimulation in mice. Life Sci. 83, 2375–2381. Meldrum, B., Garthwaite, J., 1990. Excitatory amino acid neurotoxicity

Grebb, J.A., Ellsworth, K.A., Freed, W.J., 1985. Differences between and neurodegenerative diseases. Trends Pharmacol. Sci. 11, 379–387.calcium channel inhibitors in their effects on phencyclidine-induced Meldrum, B.S., 1995. Excitatory amino acid receptors and their role inbehavioural stimulation in mice. Pharmacol. Biochem. Behav. 23, epilepsy and cerebral ischemia. Ann. N.Y. Acad. Sci. 757, 492–506.615–618. Mena, M.A., Garcia de Yebenes, M.J., Tabernero, C., Casarejos, M.J.,

Griebel, G., 1995. 5-Hydroxytryptamine-interacting drugs in animal Pardo, B., Garcia de Yebenes, J., 1995. Effects of calcium antagonistsmodels of anxiety disorders: more than 30 years of research. Phar- on the dopamine system. Clin. Neuropharmacol. 18, 410–426.macol. Ther. 65, 319–395. Misane, I., 1993. Brain function enhancing activity of cerebrocrast, a

Hiramatsu, M., Cho, A.K., Nabeshima, T., 1989. Comparison of the novel type of the 1,4-dihydropyridine compounds. Ph.D. thesis,behavioural and biochemical effects of the NMDA receptor antago- Latvian Academy of Sciences and University of Latvia, Riga, pp.nists, MK-801 and phencyclidine. Eur. J. Pharmacol. 166, 359–366. 1–80.

Hoffmeister, F., Bellemann, H.P., Benz, W., Van den Kerckhoff, W., 1984. Morad, M., Nayler, W., Korzola, S., Schramm, M., 1988. The CalciumPsychotropic actions of nimodipine. In: Betz, E., Deck, H., Hoffmeis- Channel: Structure, Function and Implications. Springer-Verlag, Berlin.ter, F. (Eds.), Nimodipine: Pharmacological and Clinical Properties. Muceniece, R., Liepa, I., Bleidelis, E., Svirskis, S., Klusa, V., 1991.Schattauer Verlag, New York. Stress-protective action of 1,4-dihydropyridines (Abstract). In: Klusa,

Honore, T., Drejer, J., Nielsen, E.O., Watkins, J.C., Olverman, H.J., V. (Ed.) Pharmacology of Novel Centrally and Peripherally ActingNielsen, M., 1989. Molecular target size analyses of the NMDA- Drugs. Latvian Institute of Organic Synthesis, Riga, pp. 51–52.receptor complex in rat cortex. Eur. J. Pharmacol. 172, 239–247. O’Neill, S.K., Bolger, T., 1989. Phencyclidine and MK-801: behavioral

Ishimaru, M.J., Toru, M., 1997. The glutamate hypothesis of schizophre- and neurochemical comparison of their interactions with dihydro-nia. Therapeutic implications. CNS Drugs 7, 47–67. pyridine calcium antagonists. Brain Res. Bull. 22, 611–616.

¨Jackson, D.M., Johansson, C., Lindgren, L.M., Bengtsson, A., 1994. Ogren, S.O., Goldstein, M., 1994. Phencyclidine- and dizolcipine-inducedDopamine receptor antagonists block amphetamine- and hyperlocomotion are differentially mediated. Neuropsychopharmacolo-phencyclidine-induced molor stimulation in rats. Pharmacol. Biochem. gy 11, 167–177.

¨ ¨Behav. 48, 465–471. Ogren, S.O., Kohler, C., Fuxe, K., Angeby, K., 1979. Behavioural effectsJavitt, D.C., Zukin, S.R., 1991. Recent advances in the phencyclidine of ergot drugs. In: Fuxe, K., Calne, D.B. (Eds.), Dopamine Ergot

model of schizophrenia. Am. J. Psychiatry 148, 1301–1308. Derivatives and Motor Function. Pergamon Press, Oxford, pp. 187–Jensen, K., 1959. Depressions in patients treated with reserpine for 205.

arterial hypertension. Acta Psychiatr. Scand. 34, 195–204. Pop, E., Bodor, N., 1992. Chemical systems for delivery of antiepilepticJohnson, K.M., 1987. Phencyclidine: behavioural and biochemieal evi- drugs to the central nervous system. Epilepsy Res. 13, 1–16.

dence supporting a role for dopamine. Fed. Proc. 42, 2579–2583. Popoli, P., Benedetti, M., Scotti de Carolis, A., 1990. Influence of DHPJohnson, K.M., Jones, S.M., 1990. Neuropharmacology of phencyclidine: Iigands on PCP-induced effects in rats. Pharmacol. Res. 22 (Suppl. 1),

Basic mechanisms and therapeutic potential. Annu. Rev. Pharmacol. 7–8.Toxicol. 30, 717–750. Porsolt, R.D., Anton, G., Blavet, N., Jalfre, M., 1978. Behavioural despair

Karler, R., Calder, L.D., Turkanis, S.A., 1991. Calcium channel blockers in rats: a new model sensitive to antidepressant treatments. Eur. J.and excitatory amino acids. Brain Res. 551, 331–333. Pharmacol. 47, 379–391.

Karpova, M.N., Germane, S., Cebers, C.J., Pankov, O.Y., Klusha, V.E., Porsolt, R.D., Bertin, A., Blavet, N., Deniel, M., Jalfre, M., 1979.Duburs, G.Y., Bisenieks, E.A., 1993. The anticonvulsant activity of Immobility induced by forced swimming in rats: effects of agentsglutapyrone-a new type of derivative of amino acid-containing 1,4- which modify central catecholamine and serotonin activity. Eur. J.dihydropyridines. Bull. Exp. Biol. Med. 16, 1119–1121. Pharmacol. 57, 201–210.

Karpova, M.N., Germane, S.K., Pankov, O.Y., Klusha, V.E., Dubur, G.Y., Porsolt, R.D., Bertin, A., Jalfre, M., 1977. Behaviour despair in mice: a1993. Effects of glutapyrone, a new amino acid-containing 1,4- primary screening test for antidepressants. Arch. Int. Pharmacodyn.dihydropyiidine, on focal penicillin-induced epileptic activity and on Ther. 229, 327–336.


Pucilowski, O., 1992. Psychopharmacological properties of calcium Tazi, A., Farh, M., Hakkou, F., 1991. Behavioral effects of calciumchannel inhibitors. Psychopharmacology 109, 12–29. channel antagonist: Nifedipine. Fundam. Clin. Pharmacol. 5, 229–236.

Rothman, R.B., 1994. PCP site 2: a high affinity MK-801-insensitive Triggle, D.J., 1990. Calcium, calcium channels and calcium channelphencyclidine binding site. Nerotoxicol. Teratol. 16, 343–353. antagonists. Can. J. Physiol. Pharmacol. 68, 1474–1481.

Rothman, R.B., Reid, A.A., Monn, J.A., Jacobson, A.E., Rice, K.C., Utno, L.Y., Lipsberga, Z.E., Silova, A.A., Girgensone, M.Y., Bisenieks,1989. The psychotomimetic drug phencyclidine labels two high E.A., Dubur, G.Y., 1989. Cardioprotective properties of a 1,4-dihydro-affinity sites in guinea pig brain: Evidence for N-methyl-D-aspal-tate- pyridine derivative, glutapyrone, in deep hypothermia (Russian). Bull.coupled and dopamine reuptake carrier-associated phencyclidinc bind- Eksp. Biol. Med. 109, 558–561.ing sites. Mol. Pharmacol. 36, 887–896. Veveris, M., Cirule, H., 1993. Glutapyrone-induced dose- and time-

Rubin, B., Malone, M., Waugh, M., Burke, J.C., 1957. Bioassay of dependent antiarrhythmic activity. (Abstract) in: Usage, InteractionsRauwolfia roots and alkaloids. J. Pharmacol. Exp. Ther. 1211, 125– and Adverse Toxic Action of Drugs. Kaunas Medicina, Kaunas, p. 45.136. Viveros, M.P., Martin, S., Ormazabal, M.J., Alfaro, M.J., Martin, M.I.,

Sanger, D.J., 1991. Animal models of anxiety and the screening and 1996. Effects of nimodipine and nifedipine upon behavior and regionaldevelopment of novel anxiolytic drugs. In: Boulton, A., Baker., G., brain monoamines in the rat. Psychopharmacology 127, 123–132.Martin-Iversons, M.T. (Eds.), Neuromethods, vol. 19. The Humana Weischer, M.L., 1976. Eine einfache versuchsanordnung zur quantitativen

¨ ¨ ¨Press, Clifton, pp. 147–149. berteilung von motilitat und neurgurverhalten bie mausen. Psycho-Schramm, M., Thomas, G., Towart, R., Franckowiak, G., 1983. Novel pharmacology 50, 275–279.

dihydropyridines with positive ionotropic function through activation Willner, P., 1984. The validity of animal models of depression. Psycho-21of Ca channels. Nature (London) 3113, 535–537. pharmacology 83, 1–16.

Seiden, L.S., Sabol, K.E., 1993. Amphetamine effects on catecholaminesystems and behaviour. Ann. Rev. Pharmacol. Toxicol. 32, 639–677.

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