Explant-derived human dental pulp stem cells enhance differentiation and proliferation potentials.

Numerous stem cell niches are present in the different tissues and organs of the adult human body. Among these tissues, dental pulp, entrapped within the 'sealed niche' of the pulp chamber, is an extremely rich site for collecting stem cells. In this study, we demonstrate that the isolation of human dental pulp stem cells by the explants culture method (hD-DPSCs) allows the recovery of a population of dental mesenchymal stem cells that exhibit an elevated proliferation potential. Moreover, we highlight that hD-DPSCs are not only capable of differentiating into osteoblasts and chondrocytes but are also able to switch their genetic programme when co-cultured with murine myoblasts. High levels of MyoD expression were detected, indicating that muscle-specific genes in dental pulp cells can be turned on through myogenic fusion, confirming thus their multipotency. A perivascular niche may be the potential source of hD-DPSCs, as suggested by the consistent Ca(2+) release from these cells in response to endothelin-1 (ET-1) treatment, which is also able to significantly increase cell proliferation. Moreover, response to ET-1 has been found to be superior in hD-DPSCs than in DPSCs, probably due to the isolation method that promotes release of stem/progenitor cells from perivascular structures. The ability to isolate, expand and direct the differentiation of hD-DPSCs into several lineages, mainly towards myogenesis, offers an opportunity for the study of events associated with cell commitment and differentiation. Therefore, hD-DPSCs display enhanced differentiation abilities when compared to DPSCs, and this might be of relevance for their use in therapy.

Subclassification of the presynaptic alpha 2-autoreceptors in rabbit brain cortex.

1. alpha 2-Adrenoceptor binding sites have been subclassified into alpha 2A sites of which a main characteristic is very low affinity for prazosin, and alpha 2B sites with relatively high affinity for prazosin. The presynaptic alpha 2-autoreceptors in rabbit brain cortex were studied in order to classify them in terms of alpha 2A and alpha 2B. Release of [3H]-noradrenaline in cortical slices was elicited by trains of 4 pulses delivered at 100 Hz. 2. Clonidine caused concentration-dependent inhibition of the stimulation-evoked overflow of tritium, with an EC50 of 7.5 nM and a maximal inhibition by 96%. 3. The following alpha-adrenoceptor antagonists shifted the concentration-response curve of clonidine to the right (antagonist-receptor dissociation constants KD in brackets): yohimbine (14 nM), 2-[2H-(1-methyl-1,3-dihydroisoindole)methyl]-4,5-dihydroimidazo le (BRL 44408; 15 nM) and 1,2-dimethyl-2,3,9,13betetrahydro-1H-dibenzo[c,f]imidazo[1,5-a]aze pine (BRL 41992; 630 nM). Prazosin 1 microM and 2-[2-[4-(o-methoxyphenyl)piperazine-1-yl]-ethyl]-4,4-dimethyl-1,3 (2H,4H)-isoquinolinedione (AR-C 239) 1 microM failed to antagonize the effect of clonidine. Higher concentrations of prazosin and AR-C 239 greatly accelerated the basal efflux of tritium. 4. The method used permits the functional determination of antagonist affinities undistorted by endogenous alpha 2-autoinhibition. A comparison with affinities derived from radioligand binding experiments indicates that the presynaptic alpha 2-autoreceptors in rabbit brain cortex are markedly different from the alpha 2B-subtype and probably belong to the prazosin-insensitive alpha 2A-subtype.

Ionotropic glutamate receptor types leading to adenosine-mediated inhibition of electrically evoked [3H]-noradrenaline release in rabbit brain cortex slices.

1. Glutamate inhibits the electrically evoked release of noradrenaline in rabbit brain cortex slices; the inhibition is mediated by adenyl compounds, presumably adenosine. The aim of the present study was to identify the receptors involved in this indirect inhibitory effect of glutamate. Slices of the occipitoparietal cortex were preincubated with [3H]-noradrenaline and then superfused and stimulated by trains of 6 pulses, 100 Hz. 2. The ionotropic glutamate receptor agonists alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AM-PA; 10-100 microM), kainate (10-100 microM) and N-methyl-D-aspartate (NMDA; 30-300 microM) but not the metabotropic glutamate receptor agonist, 1-amino-1,3-cyclopentanedicarboxylate (ACPD; 10-100 microM) reduced the electrically evoked overflow of tritium. 3. The effects of AMPA, kainate and NMDA were attenuated or abolished by the adenosine A1-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) as well as by adenosine A1-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) as well as by adenosine deaminase but not by the alpha 2-adrenoceptor antagonist yohimbine, the gamma-aminobutyric acid (GABA) receptor antagonists, bicuculline and 2-hydroxysaclofen and the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME). 4. The NMDA receptor antagonist, 2-amino-5-phosphonopentanoate (AP5) blocked the inhibitory effect of NMDA but not that of AMPA and kainate. The non-NMDA-receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) blocked the effect of AMPA but not of kainate and NMDA. 5. In addition to decreasing the electrically evoked overflow of tritium, AMPA, kainate and NMDA but not ACPD caused a steep but transient rise of basal tritium efflux. This immediate releasing effect was not significantly changed by DPCPX, adenosine deaminase, yohimbine, bicuculline, 2-hydroxysaclofen and L-NAME (except that L-NAME enhanced the effect of kainate). AP5 and CNQX antagonized the immediate releasing effects in the same way that they antagonized the inhibition by AMPA, kainate and NMDA of the electrically evoked overflow of tritium.6. It is concluded that AMPA, kainate and NMDA, like glutamate, reduce the electrically evoked release of noradrenaline by releasing adenosine or an adenine nucleotide which is then degraded to adenosine. Activation of each of the three ionotropic glutamate receptors, AMPA, kainate and NMDA receptors, but not activation of metabotropic glutamate receptors can initiate this indirect inhibitory effect on the release of noradrenaline (as well as the known noradrenaline releasing effect).

Evidence for P2-purinoceptor-mediated inhibition of noradrenaline release in rat brain cortex.

1. Some postganglionic sympathetic axons possess P2Y-like P2-purinoceptors which, when activated, decrease the release of noradrenaline. We examined the question of whether such receptors also occur at the noradrenergic axons in the rat brain cortex. Slices of the brain cortex were preincubated with [3H]-noradrenaline, then superfused with medium containing desipramine (1 microM) and stimulated electrically, in most experiments by trains of 4 pulses/100 Hz. 2. The selective adenosine A1-receptor agonist, N6-cyclopentyl-adenosine (CPA; 0.03-3 microM) as well as the non-subtype-selective agonist 5'-N-ethylcarboxamido-adenosine (NECA; 0.3-3 microM) reduced the evoked overflow of tritium, whereas the adenosine A2a-receptor agonist, 2-p-(2-carbonylethyl)-phenethylamino-5'-N-ethylcarboxamido-a denosine (CGS-21680; 0.003-30 microM) and the adenosine A3-receptor agonist N6-2-(4-aminophenyl)ethyl-adenosine (APNEA; 0.03-3 microM) caused no change. Of the nucleotides tested, ATP (30-300 microM), adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S; 30-300 microM), adenosine-5'-O-(2-thiodiphosphate) (ADP beta S; 30-300 microM), P1,P4-di(adenosine-5')-tetraphosphate (Ap4A; 30-300 microM) and the preferential P2Y-purinoceptor agonist, 2-methylthio-ATP (300 microM) decreased the evoked overflow of tritium. The P2X-purinoceptor agonist, alpha,beta-methylene-ATP (3-300 microM) caused no change. 3. The A1-selective antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 10 nM) attenuated the effects of the nucleosides CPA (apparent pKB value 9.8) and NECA as well as of the nucleotides ATP (apparent pKB 9.3), ATP gamma S (apparent pKB 9.2) and ADP beta S (apparent pKB 8.7). CGS-21680 and APNEA were ineffective also in the presence of DPCPX. The A2-selective antagonist 1,3-dipropyl-8-(3,4-dimethoxystyryl)-7-methylxanthine (KF-17837) reduced the effects of CPA, NECA and ATP gamma S only when given at a concentration of 300 nM but not at 1O nM.4. The P2-purinoceptor antagonists, suramin (300 micro M), reactive blue 2 (30 micro M) and cibacron blue 3GA(30 micro M) did not change the effect of CPA. Suramin and cibacron blue 3GA shifted the concentration response curve of ATP gamma S to the right (apparent pKB values 3.7 and 5.0, respectively). Reactive blue 2 also attenuated the effect of ATPyS, and cibacron blue 3GA attenuated the effect of ATP, but in these cases the agonist concentration-response curves were not shifted to the right. There was no antagonistic effect of suramin against ATP and ADP beta S.5. The results indicate that rat cerebrocortical noradrenergic axons possess, in addition to the knownadenosine Al-receptor, a separate purinoceptor for nucleotides (P2) which, in contrast to the Al-receptor,is blocked by suramin, reactive blue 2 and cibacron blue 3GA. Nucleotides such as ATP and ATP gamma S activate both receptors. Inconsistencies in antagonist effects against nucleotides are probably due to this activation of two receptors. The presynaptic P2-purinoceptor is P2Y-like, as it is in the peripheral sympathetic nervous system.

Presynaptic alpha 2-adrenoceptor, opioid kappa-receptor and adenosine A1-receptor interactions on noradrenaline release in rabbit brain cortex.

The interaction of presynaptic, release-inhibiting alpha 2-adrenoceptors, opioid kappa-receptors and adenosine A1-receptors was studied in slices of the occipito-parietal cortex of the rabbit. The slices were preincubated with 3H-noradrenaline and then superfused and stimulated electrically twice for 2 min each (S1, S2). The stimulation-evoked overflow of tritium was taken to reflect action potential-evoked release of noradrenaline. One of two release-modulating compounds to be examined for interaction was kept in the medium throughout superfusion, the other one was added before S2. In many experiments, the stimulation parameters were adjusted (frequency 0.5-7 Hz; voltage drop 2-5 V/cm) in order to obtain similar reference release (S1) values despite the presence of the first release-modulating compound. The selective kappa-receptor agonist ethylketocyclazocine (EK) attenuated markedly the release-inhibiting effects of the alpha 2-adrenoceptor-selective agonists clonidine and alpha-methylnoradrenaline as well as the release-facilitating effect of the alpha 2-adrenoceptor-selective antagonist yohimbine. The attenuation occurred both when the parameters of electrical stimulation were kept constant and when they were adjusted to obtain similar S1 release values. The selective A1-receptor agonist R-N6-phenylisopropyladenosine (PIA) also attenuated the effects of clonidine and yohimbine. Conversely, clonidine attenuated and yohimbine enhanced the release-inhibiting effect of PIA. Yohimbine also enhanced the release-facilitating effect of the adenosine receptor antagonist 8-phenyltheophylline. Again, these changes occurred both at constant stimulation parameters and when stimulation parameters were adjusted.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of verapamil, diltiazem and ryosidine on the release of dopamine and acetylcholine in rabbit caudate nucleus slices.

Slices of the rabbit caudate nucleus were preincubated with 3H-dopamine or 3H-choline and then superfused with label-free medium. Release of 3H-dopamine and 3H-acetylcholine was elicited by either electrical stimulation at 8 (in one series 2) Hz, or an increase in the K+ concentration by 50 mmol/l, or addition of L-glutamate 1 mmol/l. Verapamil 1 mumol/l, diltiazem 1 and 10 mumol/l, and ryosidine 1 mumol/l failed to the reduce the electrically-, K+- and glutamate-evoked overflow of tritium. Verapamil 1 mumol/l and diltiazem 10 mumol/l also failed to reduce the electrically-evoked overflow (2 Hz) when dopamine receptors, neuronal dopamine uptake, and neuronal choline uptake were blocked by domperidone, nomifensine and hemicholinium, respectively. Inhibition of the evoked overflow of tritium was only obtained when concentrations were increased to verapamil 10 mumol/l, diltiazem 100 mumol/l and ryosidine 10 mumol/l. The inhibition was generally small. It was more evident for slices preincubated with 3H-choline than for those preincubated with 3H-dopamine, because in the latter verapamil, diltiazem and (much less) ryosidine accelerated the basal efflux of tritium. The inhibition of the K+-evoked overflow of tritium was probably due to blockade of Ca2+ channels because this overflow was Ca2+-dependent but tetrodotoxin-resistant. In contrast, the inhibition of the electrically- and glutamate-evoked overflow possibly involved blockade of Na+ channels as well. The results indicate that three calcium antagonists from different chemical classes are very weak inhibitors of Ca2+ entry into, and hence transmitter release from, the terminal axons of central dopaminergic and cholinergic neurones. The function of the high affinity calcium antagonist binding sites that have been identified in brain remains unknown.

Stable adenine nucleotides inhibit [3H]-noradrenaline release in rabbit brain cortex slices by direct action at presynaptic adenosine A1-receptors.

Effects of adenosine and nucleotides on the release of previously stored [3H]-noradrenaline were studied in rabbit brain cortex slices. The slices were stimulated twice, in most experiments by 6 electrical field pulses delivered at 100 Hz. Adenosine and the nucleotides AMP, ADP, ATP, AMPS, ADP beta S, ATP gamma S, beta,gamma-imido-ATP and beta,gamma-methylene-ATP all reduced the evoked overflow of tritiated compounds. For purines for which concentration-response curves were determined, the order of potency was adenosine greater than ATP approximately ATP gamma S approximately beta,gamma-imido-ATP approximately ADP greater than beta,gamma-methylene-ATP. AMP 30 mumol/l and AMPS 30 mumol/l were approximately equieffective with 30 mumol/l of adenosine and ATP gamma S, and ADP beta S 30 mumol/l was approximately equieffective with 30 mumol/l of ADP. alpha,beta-Methylene-ADP, 2-methylthio-ATP, UTP and GTP gamma S did not change the evoked overflow of tritium. alpha,beta-Methylene-ATP caused an increase; however, the increase was small and became significant only after 59 min of exposure to alpha,beta-methylene-ATP or when the slices were stimulated by 30 pulses, 10 Hz. Neither adenosine deaminase (100 U/l) nor the blocker of 5'-nucleotidase, alpha,beta-methylene-ADP (10 mumol/l), attenuated the inhibition caused by ATP, ATP gamma S and beta,gamma-methylene-ATP, despite the fact that adenosine deaminase abolished the effect of adenosine. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX, 10 nmol/l) shifted the concentration-response curves of adenosine, ATP gamma S, beta,gamma-imido-ATP and beta,gamma-methylene-ATP to the right by very similar degrees. 8-(p-Sulphophenyl)-theophylline (30 and 300 mumol/l) also markedly antagonized the inhibition produced by ATP gamma S. alpha,beta-Methylene-ATP (10 and 30 mumol/l) and suramin (100 mumol/l) did not modify the effects of adenosine, ATP gamma S and beta,gamma-methylene-ATP. It is concluded that nucleotides themselves can inhibit the release of noradrenaline in the rabbit brain cortex. The nucleotides and adenosine seem to act at the same site, i.e., the A1 subtype of the P1-purinoceptor. The results support the notion that metabolically stable, phosphate chain-modified nucleotides such as ATP gamma S, beta,gamma-imido-ATP and beta,gamma-methylene-ATP can be potent P1 agonists. No evidence was found for presynaptic P2x-, P2y- or P3-purinoceptors.

Mutual interaction between presynaptic alpha 2-adrenoceptors and opioid kappa-receptors at the noradrenergic axons of rabbit brain cortex.

The interaction between presynaptic, release-inhibiting alpha 2-adrenoceptors and opioid receptors was studied in slices of the parieto-occipital cortex of rabbits. The slices were preincubated with 3H-noradrenaline and then superfused with 3H-noradrenaline-free medium and stimulated electrically (3 or 7 Hz, 2 or 5 V/cm voltage drop between the electrodes). Clonidine and ethylketocyclazocine (EK) depressed, whereas yohimbine increased the electrically evoked overflow of tritium. When clonidine was administered first and retained in the medium for the rest of the experiment, the overflow-inhibiting effect of EK was reduced. When yohimbine was administered first and kept for the rest of the experiment, the effect of EK was enhanced. When, finally, EK was administered first and clonidine as the second drug, the overflow-inhibiting effect of clonidine was attenuated. The changes in the effect of EK (by clonidine or yohimbine) and clonidine (by EK) were not due to the changes in release per se produced by the drugs that were given first. Naloxone shifted the concentration-response curve of EK to the right; the dissociation constant of the naloxone-receptor complex, calculated from the shift, was 13 nmol/l. It is concluded that there is an interaction between presynaptic alpha 2-adrenoceptors and opioid kappa-receptors, either at the level of the receptors themselves or of the post-receptor reaction chains. Activation of one kind of receptor blunts the inhibition of release produced by activation of the other kind of receptor.

Adenosine but not an adenine nucleotide mediates tonic purinergic inhibition, as well as inhibition by glutamate, of noradrenaline release in rabbit brain cortex slices.

A possible contribution of adenine nucleotides to the endogenous purinergic, A1-receptor-mediated inhibition of noradrenaline release was studied in rabbit occipito-parietal cortex slices. The slices were preincubated with [3H]-noradrenaline and then superfused and stimulated electrically, in most experiments by trains of 6 pulses/100 Hz. A few experiments were carried out in rat occipito-parietal cortex slices. The A1-purinoceptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 1-100 nmol/l) as well as the enzyme adenosine deaminase (0.1-10 U/ml) increased the electrically evoked overflow of tritiated compounds. The maximal increase was by about 85% for both DPCPX and adenosine deaminase. The increases obtained with maximally effective concentrations of DPCPX and adenosine deaminase were not additive. The alpha 1-adrenoceptor-selective agonist methoxamine (10 but not 1 mumol/l) reduced the evoked overflow. Its effect was antagonized by yohimbine 1 mumol/l but then not attenuated further by DPCPX 100 nmol/l. L-Glutamate (300 mumol/l-2.3 mmol/l) also reduced the evoked overflow of tritium. Its effect was not changed by yohimbine 1 mumol/l but greatly, and to the same extent, attenuated by DPCPX 100 nmol/l and adenosine deaminase 3 U/ml. Neither the N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine nor omission of Mg++ changed the inhibition by glutamate. Glutamate did not alter the basal efflux of tritium from rabbit cortex slices under any experimental condition. In contrast, glutamate (100 mumol/l and 1 mmol/l) caused an immediate, marked and transient acceleration of tritium outflow from rat occipitoparietal cortex slices (medium without Mg++).(ABSTRACT TRUNCATED AT 250 WORDS)

Blockade of alpha 2-adrenoceptors permits the operation of otherwise silent opioid kappa-receptors at the sympathetic axons of rabbit jejunal arteries.

We sought for presynaptic, release-inhibiting opioid receptors at the postganglionic sympathetic axons innervating the jejunal arteries of rabbits. Evoked excitatory junction potentials (e.j.p.s; trains of 15 pulses at 1 Hz) as well as evoked overflow of tritium after preincubation with [3H]-noradrenaline (trains of 120 pulses at 1 Hz) were used to estimate transmitter release. In otherwise untreated tissues ethylketocyclazocine reduced neither the e.j.p. amplitudes nor the evoked overflow of tritium; [Met5]-enkephalin depressed the evoked overflow of tritium. Ethylketocyclazocine reduced e.j.p. amplitudes, however, in tissues exposed to either yohimbine, tolazoline or phentolamine, but not in tissues exposed to prazosin. Ethylketocyclazocine also depressed the evoked overflow of tritium when yohimbine was present. The inhibition produced by ethylketocyclazocine in the presence of yohimbine was antagonized by (-)-3-furylmethyl)-alpha-noretazocine (MR 2266) but not by N,N-diallyl-Tyr-alpha-aminoisobutyric acid-alpha-aminoisobutyric acid-Phe-Leu-OH (ICI 174864). It is concluded that the sympathetic neurones of rabbit jejunal arteries possess presynaptic kappa-receptors in addition to the previously identified delta-receptors. The kappa-receptors become operative only when presynaptic alpha 2-adrenoceptors have been blocked.

Autoreceptors and alpha 2-adrenoceptors at the serotonergic axons of rabbit brain cortex.

Slices of the rabbit occipito-parietal cortex were preincubated with 3H-serotonin and then superfused and stimulated electrically (2 min at 3 Hz). In the absence of drugs, the stimulation-evoked overflow of tritium was approximately 3% of the tritium content of the tissue. Unlabelled serotonin and 5-carboxamido-tryptamine, when administered in the presence of 6-nitroquipazine, reduced the evoked overflow of tritium. Their effects were antagonized by metitepin (apparent pA2 value 8.1) and (+/-)-cyanopindolol (apparent pA2 value 6.4). Metitepin, but not cyanopindolol, increased evoked tritium overflow; the effect of metitepin was greater in the presence than in the absence of nitroquipazine. The evoked overflow of tritium was also depressed by clonidine, an effect antagonized by idazoxan (apparent pA2 value 7.0) but not by prazosin. Phenylephrine caused a decrease only at high concentrations that simultaneously accelerated basal tritium efflux. Prazosin and idazoxan did not change evoked tritium overflow, and phentolamine increased it significantly only when administered in the presence of (+)-oxaprotiline. Rauwolscine produced an inhibition that was prevented by metitepin. It is concluded that the serotonergic axons of the rabbit occipitoparietal cortex possess presynaptic, release-inhibiting serotonin autoreceptors and alpha 2-adrenoceptors. The receptors appear to receive an input of endogenous serotonin and, to a lesser extent, noradrenaline, under the conditions of these in vitro experiments.

Acetylcholine release in rat nucleus accumbens is regulated through dopamine D2-receptors.

Experiments in slices of rat nucleus accumbens were carried out in order to investigate whether the release of acetylcholine in this tissue is modulated through dopamine receptors. The slices were preincubated with 3H-choline and then superfused and stimulated electrically twice for 2 min each at a frequency of 3 Hz. The electrically evoked overflow of tritium averaged 2.9-3.9% of the tritium content of the tissue in the various groups. The D2-selective agonist quinpirole (0.01-1 mumol/l) reduced the evoked overflow of tritium by maximally 56%, an effect antagonized by the D2-selective antagonist (-)-sulpiride (1 mumol/l). The D1-selective agonist 2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine (SKF 38393) caused a slight decrease only at the high concentration of 10 mumol/l. (-)-Sulpiride (0.1-10 mumol/l) moderately increased the evoked overflow of tritium when given alone. The dopamine uptake inhibitor nomifensine (10 mumol/l) caused a decrease, and in its presence the increase produced by (-)-sulpiride became much more marked, amounting to maximally 149% (+)-Sulpiride (0.1-1 mumol/l) failed to change the evoked overflow of tritium in the presence of nomifensine. The dopamine-releasing agent (+/-)-amphetamine (1 mumol/l) also reduced the evoked overflow, an effect abolished by (-)-sulpiride. Finally, bretylium (1 mmol/l), which blocks the release of dopamine, increased the evoked overflow. (-)-Sulpiride (1 mumol/l) lost its facilitatory effect in slices treated with bretylium. We conclude that the release of acetylcholine in rat nucleus accumbens, like its release in the nucleus caudatusputamen, is modulated through dopamine D2-receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Further functional in vitro comparison of pre- and postsynaptic dopamine receptors in the rabbit caudate nucleus.

Slices of the rabbit caudate nucleus were preincubated with 3H-dopamine or 3H-choline and then superfused and stimulated electrically. DiPr-5,6-ADTN reduced the stimulation-evoked overflow of tritium over the same concentration range, independently of whether slices had been preincubated with 3H-dopamine or 3H-choline, and the same was true for apomorphine, NPA and pergolide. Three other putative dopamine receptor agonists, namely 3-PPP, DPI and SKF 38393, failed to decrease the evoked overflow of tritium. Each of six antagonists--(-)-sulpiride, (+)-sulpiride, CGP 11109 A, cis-flupentixol, domperidone and corynanthine--increased the evoked overflow over the same concentration range in experiments with 3H-dopamine and in those with 3H-choline. For each of these antagonists except cis-flupentixol, and also for chlorpromazine, haloperidol and rauwolscine, the pA2 values against apomorphine obtained in 3H-dopamine and in 3H-choline experiments were closely similar. The antagonist effect of cis-flupentixol against apomorphine was not purely competitive. (-)-Sulpiride was a more potent antagonist than (+)-sulpiride, and cis-flupentixol was more potent than trans-flupentixol. This study supplements a previous one in which (+/-)-sulpiride, metoclopramide and molindone were used as antagonists. It is a functional in vitro approach to receptor characterization, as opposed to radioligand binding studies or in vivo investigations. The results show that a large number of dopamine receptor agonists and antagonists are unable to distinguish between the presynaptic, release-inhibiting dopamine autoreceptors and those postsynaptic dopamine receptors which, when activated, depress the release of acetylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)

A search for receptors modulating the release of gamma-[3H]aminobutyric acid in rabbit caudate nucleus slices.

Various putative striatal transmitters and related compounds were studied for their effects on the release of gamma-aminobutyric acid (GABA) from slices of the head of the rabbit caudate nucleus. The slices were preincubated with [3H]GABA and then superfused and stimulated electrically at 5 or 20 Hz. Aminooxyacetic acid was present throughout. The main changes observed were the following. The basal and, less consistently, the electrically evoked overflow of [3H]GABA were enhanced by 3,4-dihydroxyphenylethylamine (dopamine), an effect not blocked by cis-flupentixol or domperidone and not mimicked by apomorphine and D1-selective agonists. The electrically evoked overflow was diminished by 5-hydroxytryptamine (serotonin); the inhibition was prevented by methiothepin. The basal but not the electrically evoked overflow was enhanced by carbachol; acetylcholine and nicotine also accelerated the basal outflow whereas oxotremorine caused no consistent change; the effect of carbachol and acetylcholine were blocked by hexamethonium but not by atropine or by tetrodotoxin. These findings indicate that the GABA neurons in the caudate nucleus may be stimulated by dopamine, although the receptor type involved remains unclear; inhibited by serotonin; and stimulated by acetylcholine acting via a nicotine receptor. However, all drug effects observed were relatively small. No evidence was obtained for autoreceptors, alpha 2-adrenoceptors or receptors for opioids, adenosine or substance P at the GABA neurons.

Release of previously incorporated gamma-[3H]aminobutyric acid in rabbit caudate nucleus slices.

The release of gamma-aminobutyric acid (GABA) was studied in slices of the head of the rabbit caudate nucleus. The slices were preincubated with [3H]GABA and then superfused. Aminooxyacetic acid was present throughout. Both the tritium in the slices and that in the superfusate consisted practically entirely of [3H]GABA. Stimulation for 2 min by electrical field pulses of 3 ms width and 9 V/cm voltage drop (36 mA current strength) at 5 or 20 Hz elicited an overflow of [3H]GABA that amounted to 0.23 or 0.47% of the tritium content of the tissue, respectively, and was diminished by 85% in the presence of tetrodotoxin. At higher current strength, less of the stimulation-evoked overflow was tetrodotoxin-sensitive. cis-1,3-Aminocyclohexane carboxylic acid diminished the uptake of [3H]GABA into the tissue but did not change the percentage released by electrical stimulation. Ca2+ withdrawal greatly accelerated basal [3H]GABA efflux and almost abolished the response to stimulation. Nipecotic acid 10-1,000 microM enhanced both the basal and (up to eightfold) the stimulation-evoked overflow. The method described allows us to elicit electrically a quasiphysiological, i.e., Ca2+-dependent and tetrodotoxin-sensitive, neuronal release of [3H]GABA. Nipecotic acid diverts released [3H]GABA from reuptake to overflow.