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 pH]-noradrenaline, then superfused with medium containing desipramine (1 gM) and stimulated electrically, in most experiments by trains of 4 pulses/100 Hz.2 The selective adenosine Al-receptor agonist, N6-cyclopentyl-adenosine (CPA; 0.03-3 gM) as well as the non-subtype-selective agonist 5'-N-ethylcarboxamido-adenosine (NECA; 0.3-3 EM) reduced the evoked overflow of tritium, whereas the adenosine A2.-receptor agonist, 2-p-(2-carbonylethyl)-phenethylamino-5'-N-ethylcarboxamido-adenosine (CGS-21680; 0.003-30;LM) and the adenosine A3-receptor agonist N6-2-(4-aminophenyl)ethyl-adenosine (APNEA; 0.03-31M) caused no change. Of the nucleotides tested, ATP (30-300 JM), adenosine-5'-O-(3-thiotriphosphate) (ATPyS; 30-300 pM), adenosine-5'-O-(2-thiodiphosphate) (ADPPS; 30-300ILM), P1,P4-di(adenosine-5')-tetraphosphate (Ap4A; 30-3001LM) and the preferential P2Y-purinoceptor agonist, 2-methylthio-ATP (300 tM) decreased the evoked overflow of tritium. The P' -purinoceptor agonist, a,-methylene-ATP (3-3001fM) caused no change.3 The Al-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), ATPyS (apparent pKB 9.2) and ADPBS (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 ATPyS only when given at a concentration of 300 nM but not at 1O nM. 4 The P2-purinoceptor antagonists, suramin (300 ,M), reactive blue 2 (30 1M) and cibacron blue 3GA (301M) did not change the effect of CPA. Suramin and cibacron blue 3GA shifted the concentrationresponse curve of ATPyS 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 ADPPS. 5 The results indicate that rat cerebrocortical noradrenergic axons possess, in addition to the known adenosine 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 ATPyS 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. Keywords: Rat brain cortex; PI-purinoceptor; P2-purinoceptor; presynaptic purinoceptors; noradrenaline release; ad...
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)
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.
1 a2-Adrenoceptor binding sites have been subclassified into a2A sites of which a main characteristic is very low affinity for prazosin, and a2B sites with relatively high affinity for prazosin. The presynaptic a2-autoreceptors in rabbit brain cortex were studied in order to classify them in terms of a2A and a2B* 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%.
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