L. Späth

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

SummarySlices 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. Both receptors can be classified as D2. There was an excellent correlation between pA2 values and the — log Ki values of antagonists, taken from the literature, for inhibition of the binding of 3H-spiperone to rat striatal membrane fragments. The correlation supports the view that the sites labelled by 3H-spiperone are true receptors, although the affinities in the binding experiments were consistently lower than the functionally determined affinities in the intact tissue.

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 μm) 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 μm) as well as the non‐subtype‐selective agonist 5′‐N‐ethylcarboxamido‐adenosine (NECA; 0.3‐3 μm) reduced the evoked overflow of tritium, whereas the adenosine A2a‐receptor agonist, 2‐p‐(2‐carbonylethyl)‐phenethylamino‐5′‐N‐ethylcarboxamido‐adenosine (CGS‐21680; 0.003–30 μm) and the adenosine A3‐receptor agonist N6‐2‐(4‐aminophenyl)ethyl‐adenosine (APNEA; 0.03‐3 μm) caused no change. Of the nucleotides tested, ATP (30–300 μm), adenosine‐5′‐0‐(3‐thiotriphosphate) (ATP7S; 30–300 μm), adenosine‐5′‐0‐(2‐thiodiphosphate) (ADPγS; 30–300 μm), P1, P4‐di(adenosine‐5−)‐tetraphosphate (Ap4A; 30–300 μm) and the preferential P2Y‐purinoceptor agonist, 2‐methylthio‐ATP (300 μm) decreased the evoked overflow of tritium. The P2X‐purinoceptor agonist, α,β‐methylene‐ATP (3–300 μm) 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), ATP7S (apparent pKB 9.2) and ADPβS (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 ATPγS only when given at a concentration of 300 nm but not at 10 nm. 4 The P2‐purinoceptor antagonists, suramin (300 μm), reactive blue 2 (30 μm) and cibacron blue 3GA (30 μm) did not change the effect of CPA. Suramin and cibacron blue 3GA shifted the concentration‐response curve of ATPγS to the right (apparent pKB values 3.7 and 5.0, respectively). Reactive blue 2 also attenuated the effect of ATPγS, 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 ADPβS. 5 The results indicate that rat cerebrocortical noradrenergic axons possess, in addition to the known adenosine A1‐receptor, a separate purinoceptor for nucleotides (P2) which, in contrast to the A1‐receptor, is blocked by suramin, reactive blue 2 and cibacron blue 3GA. Nucleotides such as ATP and ATPγS 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.

A Search for Receptors Modulating the Release of γ-[3H]Aminobutyric Acid in Rabbit Caudate Nucleus Slices

Abstract: Various putative striatal transmitters and related compounds were studied for their effects on the release of γ‐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 out‐flow whereas oxotremorine caused no consistent change; the effects 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, α2‐adrenoceptors or receptors for opioids, adenosine or substance P at the GABA neurons.

Presynaptic α2-adrenoceptor, opioid κ-receptor and adenosine A1-receptor interactions on noradrenaline release in rabbit brain cortex

SummaryThe interaction of presynaptic, release-inhibiting α2-adrenoceptors, opioid κ-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 κ-receptor agonist ethylketocyclazocine (EK) attenuated markedly the release-inhibiting effects of the α2-adrenoceptor-selective agonists clonidine and α-methylnoradrenaline as well as the release-facilitating effect of the α2-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. EK attenuated the release-inhibiting effect of PIA, and conversely PIA attenuated the effect of EK, both at constant and at adjusted parameters of electrical stimulation. The release-inhibiting effects of tetrodotoxin and Cd2+ remained unchanged in the presence of clonidine or EK.These results demonstrate mutual interactions between presynaptic a2-, opioid K- and adenosine A1-receptors. As soon as any one of the three systems is activated, the inhibition due to activation of either of the two remaining systems is blunted. The interactions are not a consequence of the change in release per se that the first receptor ligand inevitably produces. α-Adrenoceptors interact with opioid κ-receptors in a similar manner, independently of the chemical nature (imidazoline or phenylethylamine derivative) of the α-agonist used. The interaction is specific for release-modulating receptors and does not extend to Na+ ar Ca2+ channel blockers. It may occur at the level of the receptors themselves or at the post-receptor transduction mechanisms.

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

SummaryEffects 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, ADPβS, ATPyS, β,γ-imido-ATP and β,γ-methyl-ene-ATP all reduced the evoked overflow of tritiated compounds. For purines for which concentration-response curves were determined, the order of potency was adenosine > ATP ≈ ATPyS β,γ-imido-ATP ≈ ADP > β,γ-methylene-ATP. AMP 30 Etmol/l and AMPS 30 μmol/l were approximately equieffective with 30 μmol/l of adenosine and ATPγS, and ADPβS, 30 μmol/l was approximately equieffective with 30 μmol/l of ADP. α,β-Methylene-ADP, 2-methylthio-ATP, UTP and GTPγS did not change the evoked overflow of tritium. α,β-Methylene-ATP caused an increase; however, the increase was small and became significant only after 59 min of exposure to α,β-methylene-ATP or when the slices were stimulated by 30 pulses, 10 H2. Neither adenosine deaminase (100 U/l) nor the blocker of 5′-nucleotidase, α,β-methylene-ADP (10 μmol/l), attenuated the inhibition caused by ATP, ATPyS and β,γ-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, ATPyS, β,γ-imido-ATP and β,γ-methylene-ATP to the right by very similar degrees. 8(p-Sulphophenyl)-theophylline (30 and 300 μmol/l) also markedly antagonized the inhibition produced by ATPγS. α,β-Methylene-ATP (10 and 30 μmol/l) and suramin (100 gmol/l) did not modify the effects of adenosine, ATPγS and β,γ-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 ATPγS, β,γ-imido-ATP and β,γ-methylene-ATP can be potent P1 agonists. No evidence was found for presynaptic P2X-, P2Y- or P3-purinoceptors.

Blockade of α2-adrenoceptors permits the operation of otherwise silent opioid κ-receptors at the sympathetic axons of rabbit jejunal arteries

SummaryWe 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 the 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 ethylketocylazocine 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)-α-noretazocine (MR 2266) but not by N,N-diallyl-Tyr-α-aminoisobutyric acid-α-aminoisobutyric acid-Phe-Leu-OH (ICI 174864). It is concluded that the sympathetic neurones of rabbit jejunal arteries possess presynaptic κ-receptors in addition to the previously identified δ-receptors. The κ-receptors become operative only when presynaptic α2-adrenoceptors have been blocked.

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

SummarySlices 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 μmol/l, diltiazem 1 and 10 μmol/l, and ryosidine 1 μmol/l failed to the reduce the electrically-, K+- and glutamate-evoked overflow of tritium. Verapamil 1 μmol/l and diltiazem 10 μmol/l also failed to reduce the electricallyevoked 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 μmol/l, diltiazem 100 μmol/l and ryosidine 10 μmol/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 glutamateevoked 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.

Autoreceptors and α2-adrenoceptors at the serotonergic axons of rabbit brain cortex

SummarySlices 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 α2-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.

Subclassification of the presynaptic α2‐autoreceptors in rabbit brain cortex

1 α2‐Adrenoceptor binding sites have been subclassified into α2A sites of which a main characteristic is very low affinity for prazosin, and α2B sites with relatively high affinity for prazosin. The presynaptic α2‐autoreceptors in rabbit brain cortex were studied in order to classify them in terms of α2A and α2B. 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 α‐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‐dihydroimidazole (BRL 44408; 15 nm) and 1,2‐dimethyl‐2,3,9,13b‐tetrahydro‐1H‐dibenzo[c,f]imidazo[1,5‐a]azepine (BRL 41992; 630 nm). Prazosin 1 μm and 2‐[2‐[4‐(o‐methoxyphenyl)piperazine‐1‐yl]‐ethyl]‐4,4‐dimethyl‐1,3(2H,4H)‐isoquinolinedione (AR‐C 239) μm 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 α2‐autoinhibition. A comparison with affinities derived from radioligand binding experiments indicates that the presynaptic α2‐autoreceptors in rabbit brain cortex are markedly different from the α2B‐subtype and probably belong to the prazosin‐insensitive α2A‐subtype.

Release of Previously Incorporated γ-[3H]Aminobutyric Acid in Rabbit Caudate Nucleus Slices

Abstract: The release of γ‐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 μM 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.