Effects of selective h5-HT1B (SB-216641) and h5-HT1D (BRL-15572) receptor ligands on guinea-pig and human 5-HT auto- and heteroreceptors

Human cerebral cortical slices and synaptosomes, guinea-pig cerebral cortical slices and human right atrial appendages were used to study the effects of SB-216641, a preferential h5-HT_1B receptor ligand, and of BRL-15572, a preferential h5-HT_1D receptor ligand, on the presynaptic h5-HT_1B and h5-HT_1B-like autoreceptors in the human and guinea-pig brain preparations, respectively, and on the presynaptic h5-HT_1D heteroreceptors in the human atrium. The brain preparations, preincubated with [³H]serotonin ([³H]5-HT), and the segments of atrial appendages, preincubated with [³H]noradrenaline, were superfused with modified Krebs’ solution and tritium overflow was evoked electrically (human and guinea-pig cerebral cortex slices and human atrial appendages) or by high K⁺ (human cerebral cortex synaptosomes). The electrically evoked tritium overflow from guinea-pig cerebral cortex slices was reduced by the 5-HT receptor agonist 5-carboxamidotryptamine (5-CT). This effect was not modified by BRL-15572 (2μM; concentration 154 times higher than its K_i at h5-HT_1D receptors) but was antagonized by SB-216641 (0.1μM; concentration 100 times higher than its K_i at h5-HT_1B receptors; apparent pA₂ 8.45). SB-216641 (0.1μM) by itself facilitated, whereas BRL-15572 (2μM) did not affect, the evoked overflow. In human cerebral cortex slices SB-216641 (0.1μM) also facilitated, and BRL-15572 (2μM) again failed to affect, the electrically evoked tritium overflow. In human cerebral cortical synaptosomes, 5-CT reduced the K⁺-evoked tritium overflow. This response was unaffected by BRL-15572 (300nM) but antagonized by SB-216641 (15nM; drug concentrations 23 and 15 times higher than their K_i at h5-HT_1D and h5-HT_1B receptors, respectively). Both drugs, given alone, did not modify the K⁺-evoked tritium overflow. In human atrial appendages, the electrically evoked tritium overflow was inhibited by 5-HT in a manner susceptible to antagonism by BRL-15572 (300nM; 23 times K_i at h5-HT_1D receptors) but not by SB-216641 (30nM; 30 times K_i at h5-HT_1B receptors). Both drugs by themselves did not change the electrically evoked tritium overflow. In conclusion, SB-216641 behaves as a preferential antagonist at native human 5-HT_1B receptors and BRL-15572 as a preferential antagonist at native human 5-HT_1D receptors. These compounds are clearly useful tools for the differentiation between human 5-HT_1B and 5-HT_1D receptors in functional studies. Received: 14 March 1997 / Accepted: 18 May 1997     

Evidence for neuro-effector transmission through postjunctional alpha 2-adrenoceptors in human saphenous vein.

The effects of the alpha 1-adrenoceptor antagonist prazosin and the alpha 2-adrenoceptor antagonist yohimbine were examined against stimulation-evoked contractions in human isolated saphenous veins. The concentration of yohimbine producing 30% inhibition of stimulation-evoked contractions (IC30) was 13.2 nM, whereas the IC30 of prazosin was greater than 250 nM. The inhibition of stimulation-evoked contractions by yohimbine was not prejunctionally mediated since yohimbine (0.01-0.1 microM) significantly potentiated the stimulation-evoked overflow of tritium in tissues pre-incubated with [3H]-noradrenaline. The high potency of yohimbine and the low potency of prazosin indicate that neuro-effector transmission in human saphenous vein is mediated predominantly by postjunctional alpha 2-adrenoceptors.     

Pre- and postjunctional alpha-adrenoceptor-mediated effects of prazosin, methoxamine and 6-fluoronoradrenaline in blood-perfused canine skeletal muscle in situ

Pre- and postjunctional effects of the alpha 1-selective adrenoceptor antagonist prazosin and the alpha 1- and alpha 2-selective adrenoceptor agonists methoxamine and 6-fluoronoradrenaline, respectively, were studied in skeletal muscle in situ. Prazosin reduced the vasoconstriction and enhanced the overflow of endogenous noradrenaline elicited by sympathetic nerve stimulation (1-4 Hz, 2 min); the threshold concentration was 10-100 times lower for postjunctional than for prejunctional alpha-adrenoceptor blockade. The enhancement of noradrenaline overflow by prazosin was not inversely frequency-dependent, as shown elsewhere for alpha 2-adrenoceptor antagonists. Thus, different mechanisms may be involved. Inhibition of prostaglandin synthesis by diclofenac did not alter the stimulation-evoked noradrenaline overflow, indicating a minor importance of prostaglandin-mediated transjunctional mechanisms in the modulation of noradrenaline overflow. Methoxamine and 6-fluoronoradrenaline elevated the basal vascular tone and, at higher concentrations, reduced the stimulation-evoked noradrenaline overflow. Methoxamine was 20 times more selective than 6-fluoronoradrenaline for postjunctional receptors. Our results are compatible with a pre- and postjunctional localization of alpha 2-adrenoceptors and a predominantly, but not exclusively, postjunctional localization of alpha 1-adrenoceptors. The postjunctional selectivity for prazosin was less marked than previously reported from in vitro studies. Hence, care should be taken when extrapolating in vitro findings to the more complex in vivo situation.     

Inhibition of N-methyl-d-aspartate (NMDA) and l-glutamate-induced noradrenaline and acetylcholine release in the rat brain by ethanol

Summary The influence of ethanol on stimulation-evoked ³H-transmitter release was examined in slices of the rat brain cortex and corpus striatum preincubated with ³H-noradrenaline and ³H-choline, respectively. ³H-Transmitter release was stimulated by NMDA, l-glutamate, electrical impulses, reintroduction of Ca²⁺ ions (Ca²⁺-evoked release; after superfusion with Ca²⁺-free, K⁺-rich solution) or veratridine. In cortical slices preincubated with ³H-noradrenaline and superfused with Mg²⁺-free, otherwise physiologically composed salt solution, ethanol inhibited the NMDA- or l-glutamate-induced tritium overflow (IC₅₀ 45 and 37 mmol/l, respectively). In contrast, the tritium overflow in response to electrical stimulation, reintroduction of Ca²⁺ ions or veratridine was not affected by ethanol at concentrations up to 320 mmol/l; these experiments were carried out in cortical slices superfused with solution containing a physiological Mg²⁺ concentration. Ethanol also failed to inhibit Ca²⁺-evoked release in the absence of Mg2⁺ ions. In the presence of 1 mol/l veratridine, but not in its absence, NMDA induced tritium overflow even when cortical slices were superfused with salt solution containing a physiological Mg²⁺ concentration; again, ethanol inhibited this NMDA-evoked tritium overflow (IC₅₀ 73 mmol/l). In striatal slices preincubated with ³H-choline and superfused with Mg²⁺-free physiological salt solution, the NMDA-evoked tritium overflow was also, although at lower potency, inhibited by ethanol (IC₅₀ 192 mmol/l).In spite of the differences between the IC₅₀ values of ethanol determined for the inhibition of cortical noradrenaline and striatal acetylcholine release, it may be concluded that the NMDA receptor-ion channel complex is one of the sites of action underlying the ethanol-induced inhibition of neurotransmitter release. Since in the brain cortex the NMDA-induced ³H-noradrenaline release appears to be mediated by an excitatory interneurone activated by NMDA, this neuronal system may be involved in the cortical actions of ethanol.     

Involvement of alpha-receptors in clonidine-induced inhibition of transmitter release from central monoamine neurones

Slices of rat cerebral cortex preincubated with (?)-³H-noradrenaline or ³H-5-hydroxytryptamine were stimulated by an electrical field, and the stimulation-induced overflow of tritium was determined. (1) Clonidine diminished the stimulation-evoked tritium overflow from slices preincubated with ³H-noradrenaline. The degree of this inhibition was greater at a low than at a high frequency of stimulation. (2) A high concentration of clonidine (10^?5 M) did not antagonize the increase of the stimulation-induced overflow caused by 10^?6 M or 10^?5 M cocaine, but abolished the increase caused by 10^?7 M of phentolamine or phenoxybenzamine. In the presence of cocaine, the inhibitory effect of clonidine was reduced. (3) 10^?5 M clonidine diminished the stimulation-evoked overflow of tritium from slices preincubated with ³H-5-hydroxytryptamine. (4) It is concluded that clonidine decreases, and phentolamine and phenoxybenzamine increase, the stimulation-induced release of noradrenaline from cerebral neurones by an activation and a blockade of α-receptors, respectively. A variety of secretory cells (secreting catecholamines, acetylcholine, 5-hydroxytryptamine, insulin or renin) seem to be endowed with structures similar to α-adrenergic receptors, which can modulate the secretion process.     

Opioid receptor sub-types involved in the control of transmitter release in cortex of the brain of the rat

Electrical stimulation (3 Hz, 2msec duration, 5–12 V for 2 min every 20min) of cortical slices from the rat, previously incubated with [³H]noradrenaline, evoked a release of tritium which was inhibited by morphine, normorphine, Tyr-d-Ala-Gly-MePhe-NH(CH₂)₂OH (RX783006) and d-Ala²-d-Leu⁵-enkephalin (pIC₃₀ 5.90, 6.32. 7.45 and 6.74 respectively). Naloxone did not affect the release of tritium when given alone but antagonised the actions of the opioids, giving a K_e value of about 3 nM irrespective of the particular agonist used, which suggests an action at mu receptors. The delta opioid receptor blocker, ICI154129, antagonised the opioids only in large concentrations (K_e 21300nM). In slices previously incubated with [³H]5-hydroxytryptamine, electrical stimulation increased overflow of tritium but neither naloxone nor the opioid agonists affected evoked overflow of tritium at concentrations which were effective in slices incubated with [³H]noradrenaline. It is concluded that stimulation of mu opioid receptors may inhibit release of noradrenaline from central noradrenergic neurones and that these receptors are not present in significant numbers on neurones releasing 5-hydroxytryptamine in the cortex.     

Subclassification of release-regulating alpha 2-autoreceptors in human brain cortex.

1. Release-regulating alpha 2-autoreceptors in human brain were characterized pharmacologically in cortical slices from patients undergoing neurosurgery to remove subcortical tumours; the slices were prelabelled with [3H]-noradrenaline ([3H]-NA) and stimulated electrically (3 Hz, 2 ms, 24 mA) under superfusion conditions. 2. The stimulus-evoked tritium overflow was almost totally Ca(2+)-dependent and tetrodotoxin-sensitive. 3. Clonidine and oxymetazoline 0.01 to 1 microM inhibited in a concentration-dependent manner the evoked overflow of tritium. The two drugs were equipotent (EC50 = 0.03 microM) and their maximal effect was approx. 45%. Phenylephrine and methoxamine, up to 1 microM, did not affect tritium overflow. 4. Yohimbine (0.01-0.1 microM) shifted the concentration-response curve of clonidine to the right. The calculated pA2 value was 8.29. 5. Prazosin and 2-[2-[4-(o-methoxyphenyl)piperazine-1-yl]ethyl]-4,4- dimethyl-1,3(2H,4H)-isoquinolinedione (AR-C 239), tested at 0.3 microM, did not modify the concentration-response curve of clonidine. 6. The effect of clonidine was antagonized by (+)-mianserin (pA2 = 7.74), but not by up to 0.3 microM of the (-)-enantiomer. The concentration-response curve of clonidine was shifted to the right by the novel alpha 2-adrenoceptor antagonist, 5-chloro-4-(1-butyl-1,2,5,6-tetrahydropyridin-3-yl)-thiazole-2-ami ne (Z)-2-butenedioate (1:1) salt (ORG 20350) (pA2 = 7.55). 7. Yohimbine, (+)-mianserin and ORG 20350, but not prazosin and (-)-mianserin, increased the electrically-evoked tritium overflow, suggesting that autoreceptors may be tonically activated by endogenous NA. 8. Desipramine (1 microM) increased evoked tritium overflow from human cortex slices.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alpha-adrenoceptor antagonists and the release of noradrenaline in rabbit cerebral cortex slices: support for the alpha-autoreceptor hypothesis.

Slices of rabbit cerebral cortex were preincubated with [3H]-noradrenaline and then superfused and stimulated electrically twice for 2 min each (S1, S2) at various frequencies (0.2-3 Hz). The stimulation-evoked overflow of tritium (S1) increased with increasing frequency and was higher when cocaine (10 microM) was present. In the absence of cocaine, tetraethylammonium (TEA; 100 and 300 microM), added before S2, increased the stimulation-evoked overflow of tritium to about the same extent, irrespective of the frequency. In contrast, rauwolscine (0.1 and 1 microM) and idazoxan (0.1-10 microM) increased the evoked overflow much more, the higher the frequency of stimulation. Phentolamine (0.1 and 1 microM) reduced the overflow elicited at 0.3 and 1 Hz, and (1 microM) caused an increase only at 3 Hz. In slices superfused throughout with cocaine 10 microM, rauwolscine (1 microM) and idazoxan (1 and 10 microM) again increased the evoked overflow of tritium more, the higher the frequency of stimulation. For a given frequency, rauwolscine and idazoxan enhanced the evoked overflow to a greater extent in the presence than in the absence of cocaine. Idazoxan (1 and 10 microM) and rauwolscine (1 microM) counteracted the inhibition that phentolamine (0.1 microM) produced at low frequency. The increases caused by rauwolscine (1 microM) and TEA (300 microM) were approximately additive, but those caused by rauwolscine (1 microM) and idazoxan (10 microM) were not. The effects of rauwolscine, idazoxan and phentolamine depend on the experimental conditions (frequency, cocaine) in a manner compatible with the operation of a presynaptic alpha 2-adrenoceptor-mediated autoinhibition of noradrenaline release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of clonidine on the stimulation-evoked release of3H-noradrenaline from superfused rat brain slices as a function of the biophase concentration

The influence of clonidine on the stimulation-evoked overflow of tritium was studied in brain slices preincubated with 3H-noradrenaline. The slices were prepared from parietal cortex (Cx), nucleus anterior hypothalami (nah) and nucleus tractus solitarii (nts). After preincubation, the tissues were superfused at 23 degrees C or 37 degrees C with a medium containing the noradrenaline uptake inhibitor desipramine. Electrical field stimulation was applied using stimulation frequencies of 0.3-10 Hz. At 23 degrees C/0.3 Hz, clonidine concentration-dependently inhibited the evoked overflow of tritium in all three brain regions. In contrast, at 23 degrees C/3 Hz the inhibitory effect of the drug in the Cx was abolished and a facilitation was observed in the nah and nts. When tested at increasing frequencies of stimulation in the nts at 23 degrees C, clonidine exerted a dual action, characterized by a reduction of electrically evoked responses at frequencies below 1 Hz and a facilitation at frequencies above 1 Hz. At 37 degrees C, clonidine concentration-dependently decreased the evoked overflow in all brain regions studied, this effect being more pronounced at 0.3 Hz than at 3 Hz. The apparent lack of an effect of clonidine on the stimulation-evoked overflow of tritium in the Cx at 23 degrees C/3 Hz was turned to a facilitation when noradrenaline (0.01 mumol/l) was included in the superfusion medium. Conversely, an inhibitory effect of clonidine was seen when the uptake blocker desipramine (as well as noradrenaline) was omitted from the superfusion medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of noradrenaline release in human cerebral cortex mediated by N-methyl-D-aspartate (NMDA) and non-NMDA receptors.

1. Human brain cortical slices from patients undergoing neurosurgery for treatment of epilepsy resistant to antiepileptic drugs were used to identify and characterize N-methyl-D-aspartate (NMDA) and non-NMDA receptors mediating stimulation of noradrenaline release. The slices preincubated with [3H]-noradrenaline were superfused with Krebs-Henseleit solution with or without Mg2+ (1.2 mmol l-1) and were stimulated by 2-min exposure to NMDA, kainic acid or (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). 2. In slices superfused without Mg2+, NMDA induced a concentration-dependent tritium overflow. 3. The NMDA-evoked tritium overflow was almost abolished by tetrodotoxin (TTX), Mg2+ or by omission of Ca2+ from the superfusion fluid. 2-Amino-5-phosphonopentanoic acid (AP5; a competitive NMDA receptor antagonist) or dizocilpine (formerly MK-801; an antagonist at the phencyclidine receptor within the NMDA-gated ion channel) inhibited the NMDA-evoked tritium overflow. The stimulatory effect of NMDA was not significantly enhanced by glycine added to the superfusion fluid but was reduced by 7-chlorokynurenic acid (an antagonist at the glycine site coupled to the NMDA receptor). 4. In slices superfused with solution containing Mg2+, kainic acid or AMPA induced a concentration-dependent tritium overflow which was susceptible to blockade by TTX. 5. The kainic acid-evoked tritium overflow was not affected by DL-(E)-2-amino-4-methyl-5-phosphono-3-pentanoic acid (CGP37849; a competitive NMDA receptor antagonist), but was inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; an antagonist at glutamate receptors of the non-NMDA type). 6. The AMPA-evoked tritium overflow was also inhibited by CNQX.2ń     

Investigation of the subtypes of α2-adrenoceptor mediating prejunctional inhibition in rat atrium and cerebral cortex

We have investigated the subtype of α₂-adrenoceptor mediating prejunctional inhibition of neurotransmission in rat atrium in comparison with the α₂-adrenoceptor mediating prejunctional inhibition in rat cerebral cortex. In rat atrium and cerebral cortex, prejunctional α₂-adrenoceptors were investigated in terms of the ability of α₂-adrenoceptor antagonists to increase the stimulation-evoked overflow of tritium in tissues pre-incubated with [³H]-noradrenaline. The relatively non-selective α₂-adrenoceptor antagonist yohimbine and the α_2D-adrenoceptor selective antagonist BRL 44408 had potencies in rat atrium which were similar to their potencies in rat cerebral cortex. The antagonists ARC 239, HV 723, WB 4101, prazosin, chlorpromazine and abanoquil, which have low affinity for α_2D-adrenoceptors, significantly increased stimulation-evoked overflow at lower concentrations in rat atrium than rat cerebral cortex. Antagonist potency at prejunctional α₂-adrenoceptors was correlated with antagonist affinity at α₂-adrenoceptor ligand binding sites in membranes of rat kidney (α_2B) and submandibular gland (α_2D), and human recombinant α_2C-adrenoceptors labelled with [³H]yohimbine. The correlation between ligand binding sites and the functional receptor in the rat cerebral cortex was significant only for the α_2D-adrenoceptor ligand binding site (r=0.87, n=8, P<0.01) as compared to the α_2B-adrenoceptor (r=0.32, n.s.) or α_2C-adrenoceptor (r=0.12, n.s.) ligand binding sites. The correlation between ligand binding sites and the functional receptor in the rat atrium was not significant for any ligand binding site, with r=0.64, 0.68 and 0.67 for the α_2D-, the α_2B- and the α_2C-adrenoceptor ligand binding sites, respectively. It is concluded that the functional prejunctional α₂-adrenoceptor of rat cerebral cortex closely resembles the α_2D-adrenoceptor ligand binding site of rat submandibular gland, but the rat atrium may contain two subypes of prejunctional α₂-adrenoceptor, α_2D and another subtype, possibly α_2B or α_2C. Received: 15 December 1997 / Accepted: 25 February 1998     

Prejunctional alpha-adrenoceptor-mediated inhibition of norepinephrine release in blood-perfused skeletal muscle in situ

The influence of alpha-adrenoceptor antagonists on the overflow of endogenous norepinephrine (NE) and vasoconstrictor responses elicited by sympathetic nerve stimulation (1-4 Hz, 2 min) was investigated in desipramine-pretreated canine blood-perfused skeletal muscle in situ. The nonselective alpha-adrenoceptor antagonist phentolamine enhanced stimulation-evoked NE overflow and reduced vasoconstrictor responses concentration-dependently. Similar effects were obtained with phenoxybenzamine (irreversible alpha-adrenoceptor antagonist). Desipramine pretreatment attenuated the enhancement of stimulation-evoked NE overflow produced by phenoxybenzamine, indicating that phenoxybenzamine also inhibits neuronal uptake. The enhancement by phenoxybenzamine was independent of the stimulation frequency, suggesting a similar engagement of prejunctional alpha-adrenoceptor-mediated inhibition of transmitter release over the frequency range studied here. The alpha 2-selective adrenoceptor antagonist yohimbine enhanced nerve stimulation-evoked NE overflow at concentrations similar to those required to antagonize vasoconstrictor responses to exogenous NE; 10-fold higher concentrations were required, however, to antagonize nerve stimulation-induced vasoconstriction. The concept of a quantitatively important prejunctional alpha 2-adrenoceptor-mediated feedback inhibition of NE release in vivo is supported by our findings in the skeletal muscle vasculature. Postjunctional alpha 2-adrenoceptors appear to be preferentially activated by circulating catecholamines but also seem to be involved in the nervous control of vascular tone.     

Presynaptic β2-adrenoceptors on the sympathetic nerve fibres of the human saphenous vein: no evidence for involvement in adrenaline-mediated positive feedback loop regulating noradrenergic transmission

Summary Spirally cut strips of human saphenous veins preincubated with ³H-noradrenaline were superfused in the presence of corticosterone and, unless stated otherwise, of cocaine or desipramine. Tritium overflow was stimulated electrically (2 Hz). Adrenaline (in the presence of rauwolscine), isoprenaline and the preferential ₂-adrenoceptor agonist procaterol concentration-dependently increased the electrically evoked tritium overflow. Prenalterol, a -adrenoceptor agonist with moderate preference for ₁-adrenoceptors, was ineffective. The concentration-response curve of isoprenaline was shifted to the right by the nonselective -adrenoceptor antagonist propranolol and by the preferential ₂-adrenoceptor antagonist ICI 118,551, but was not affected by the ₁-selective antagonist atenolol. In experiments on strips preexposed to adrenaline 10 nmol/l (i. e. a concentration higher than that which normally occurs in vivo) for 32 min in the absence of cocaine or desipramine, the electrically evoked ³H overflow was not affected 12 and 44 min after withdrawal of adrenaline, irrespective of whether propranolol was absent or present in the superfusion fluid. — In veins incubated with ³H-adrenaline, a considerable amount of the radioactivity was accumulated. During subsequent superfusion with ³H-adrenaline-free solution, electrical stimulation induced tritium overflow in a tetrodotoxin-sensitive manner. Propranolol failed to modify the evoked tritium overflow. — It is concluded that the sympathetic nerve fibres of the human saphenous vein are endowed with facilitatory presynaptic ₂-adrenoceptors. These receptors do not seem to play a substantial role in a local adrenaline (previously taken up)-mediated positive feedback loop regulating noradrenergic transmission, at least under the present in vitro conditions.This study was supported by a grant of the Deutsche Forschungsgemeinschaft Send offprint requests to M. Göthert     

Evidence for presynaptic location of inhibitory 5-HT1D\-like autoreceptors in the guinea-pig brain cortex

The effects of 5-hydroxytryptamine (5-HT) receptor agonists and antagonists on tritium overflow evoked by high K⁺ were determined in superfused synaptosomes and slices, preincubated with [³H]5-HT, from guinea-pig brain cortex. In addition, we estimated the potencies of 5-HT receptor ligands in inhibiting specific [³H]5-HT binding (in the presence of 8-hydroxy-2(di-n-propylamino)tetralin and mesulergine to prevent binding to 5-HT_1A and 5-HT_2C sites) to guinea-pig cortical synaptosomes and membranes.5-HT receptor agonists inhibited the K⁺-evoked tritium overflow from synaptosomes and slices. In synaptosomes the rank order of potencies was 2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indole-3-yl] ethylamine (L-694,247) >5-carboxamidotryptamine (5-CT) > oxymetazoline (in the presence of idazoxan) 5-HT > sumatriptan 5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole (RU 24969). The potencies of the agonists in inhibiting tritium overflow from slices correlated with those in synaptosomes, suggesting that the same site of action is involved in both preparations. In synaptosomes the nonselective antagonist at cloned human 5-HT_1D, and 5-HT_1D receptors, methiothepin, shifted the concentration-response curve for 5-CT to the right (apparent pA₂: 7.87). In contrast, ketanserin at a concentration which should block the 5-HT_1D, but not the 5-HT_1D\, receptor did not alter the inhibitory effect of 5-CT on tritium overflow. In cortical synaptosomes and membranes, [³H]5-HT bound to a single site with high affinity. In competition experiments, 5-HT receptor agonists and antagonists inhibited specific [³H]5-HT binding. In synaptosomes the rank order was L-694,247 > methiothepin >5-CT >5-methoxytryptamine >5-HT sumatriptan oxymetazoline > RU 24969 > ketanserin > ritanserin. A very similar rank order was obtained in cerebral cortical membranes. The potencies of the 5-HT receptor agonists in inhibiting tritium overflow from synaptosomes and slices correlated with their potencies in inhibiting [³H]5-HT binding to synaptosomes and membranes.In conclusion, the 5-HT receptors mediating inhibition of 5-HT release in the guinea-pig cortex are located on the serotoninergic axon terminals and, hence, represent presynaptic inhibitory autoreceptors. The [³H]5-HT binding sites in cerebral cortical synaptosomes and membranes exhibit the pharmacological properties of 5-HT_1D receptors. The correlation between the functional responses and the binding data confirms the 5-HT_1D character of the presynaptic 5-HT autoreceptors. According to the results of the interaction experiment of ketanserin and methiothepin with 5-CT on 5-HT release, the presynaptic 5-HT autoreceptors can be subclassified as 5-HT_1D\-like.     

Evidence for beta 2-adrenoceptor-mediated facilitation of 3H-noradrenaline release from isolated guinea pig papillary muscles

The effects of beta-adrenoceptor agonists and antagonists on field-stimulated release of radioactivity from superfused guinea pig papillary muscles preincubated with 3H-noradrenaline were studied. Stimulation-evoked overflow of tritium was abolished in the absence of Ca2+ or the presence of tetrodotoxin. Isoprenaline (1 mumol/L) caused a slight facilitation of evoked overflow, whereas phentolamine (1 mumol/L) exerted a strong facilitatory action. However, when phentolamine (1 mumol/L) was present throughout superfusion, isoprenaline and the selective beta 2-adrenoceptor agonist, zinterol, caused concentration-dependent increases (half-maximal effects at 1 nmol/L). The effects of the agonists were inversely related to stimulation frequency. Furthermore, the concentration-response curve of isoprenaline was shifted to the right by the selective beta 2-adrenoceptor antagonist, ICI 118,551, but not by the selective beta 1-adrenoceptor antagonist, ICI 89,406. Schild-plot analysis revealed competitive antagonism and a pA2 value of 9.04 for ICI 118,551. Both ICI 118,551 and ICI 89,406, as well as beta-adrenoceptor antagonists with intrinsic sympathomimetic activity (pindolol and celiprolol; 1 mumol/L), had no effect on stimulation-evoked overflow of tritium (phentolamine present). It is concluded that guinea pig papillary muscles are endowed with prejunctional beta 2 adrenoceptors facilitating impulse-evoked noradrenaline release. The facilitation is markedly promoted by blockade of prejunctional alpha adrenoceptors.     

Prostanoid receptors of the EP3 subtype mediate the inhibitory effect of prostaglandin E2 on noradrenaline release in the mouse brain cortex

Mouse or rat brain cortex slices were preincubated with ³H-noradrenaline and superfused with physiological salt solution containing desipramine. We studied the effects of prostaglandin E₂ (PGE₂), prostaglandin D₂ (PGD₂) and related drugs on the electrically evoked (50 mA, 2 ms, 0.3 Hz) tritium overflow.PGE₂ inhibited the electrically evoked tritium overflow from mouse brain cortex slices; the maximum effect of PGE₂ (79010) was attenuated by the ₂-adrenoceptor agonist talipexole (to 52010) and enhanced by the ₂-adrenoceptor antagonist rauwolscine (to 92%). Rauwolscine was added to the superfusion medium in all subsequent experiments. The effect of PGE₂ was readily reversible upon withdrawal from the medium and remained constant upon prolonged exposure of the tissue to the prostanoid. Studies with EP receptor agonists, mimicking the inhibitory effect of PGE₂, showed the following potencies (pIC₅₀): sulprostone (8.22); misoprostol (8.00); PGE₂ (7.74); PGEZ (7.61); iloprost (5.86). The concentration-response curve of PGE₂ was marginally shifted to the right by the EP₁ receptor antagonist AH 6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid; apparent pA₂ 3.97) and by the TP receptor antagonist vapiprost (4.50). AH 6809, by itself, did not affect the evoked overflow whereas vapiprost increased it. PGD2 inhibited the evoked overflow at high concentrations (pIC₅₀ 4.90); this effect was not altered by the DP receptor antagonist BW A868C (3-benzyl-5-(6-carboxyhexyl)-1-(2-cyclohexyl-2hydroxyethylamino)hydantoin), which, by itself, did not affect the evoked overflow. Indometacin slightly increased the evoked overflow and tended to increase the inhibitory effect of PGE₂. PGE₂ inhibited the electrically evoked tritium overflow also in rat brain cortex slices. The maximum effect (obtained in the presence of rauwolscine) was 61%; the pIC₃₀ value was 7.67.The present study suggests that PGE₂ inhibits noradrenaline release from mouse brain cortex via EP₃ receptors and that its maximum effect is more marked in the mouse than in the rat. The inhibitory effect of PGD₂ (in the mouse brain) does not involve DP receptors and may also be related to EP₃ receptors. The EP₃ receptors interact with a2-adrenoceptors and may be activated by endogenous prostanoids.     

Release of acetylcholine and its dopaminergic control in slices from striatal grafts in the ibotenic acid-lesioned rat striatum

Summary Tritium accumulation during incubation with ³-H-choline, and the efflux as well as the electrically evoked overflow of tritium during subsequent superfusion, were investigated in slices from unilateral striatal suspension grafts 16 to 20 weeks after implantation into the previously ibotenic acid-lesioned rat striatum. Slices from non-operated animals, from striata contralateral to grafts, and from animals with acute ibotenic acid lesions of the striatum were studied in parallel. The accumulation of tritium and the overflow of tritium in response to electrical stimulation (2 min, 3 Hz) were markedly impaired in acutely lesioned striata. In graft slices, tritium accumulation and the subsequent electrically evoked overflow were greater than in slices obtained after acute lesions, but were still smaller than in non-operated animals or in the contralateral striata. The dopamine D₂-receptor agonist quinpirole inhibited the electrically evoked overflow of tritium in grafts, but only to a small extent. The D₂-receptor antagonist sulpiride increased, whereas the dopamine uptake inhibitor nomifensine and the dopamine releasing drug amphetamine decreased the evoked overflow in slices from non-operated rats and from striata contralateral to grafts, but had no significant effect in grafts. As in graft slices, the release of acetylcholine in striata from animals in which the mesostriatal dopamine pathway had been lesioned by 6-hydroxy-dopamine was not changed by sulpiride and amphetamine, and was only minimally decreased by nomifensine. Our data show that striato-striatal grafts can partly restore the impaired choline accumulation and acetylcholine release in excitotoxinlesioned striata. Functional D₂-receptors are present on graft cholinergic cells, but are not activated by endogenous dopamine under the present in vitro conditions. Send offprint requests to T. Wichmann at the above address     

Effects of prostaglandin E1 and indomethacin on the stimulation- evoked overflow of 3H-noradrenaline from guinea-pig taenia caecum

Prostaglandin E₁ (5.8 × 10^?8 M) markedly and reversibly reduced the stimulation-evoked overflow of total tritium, while it had no effects on basal outflow. Indomethacin (8.4 × 10^?6 M) increased the stimulation-evoked overflow of total tritium at low frequencies (2–5 Hz), while it had no effect at high frequencies of stimulation (more than 10 Hz). It was concluded that endogenous prostaglandin E₁ also plays a regulatory role in adrenergic inhibitory neurotransmission by inhibiting the noradrenaline release from adrenergic nerve terminals of the guinea-pig taenia caecum.     

Mutual interaction of histamine H3-receptors and α2-adrenoceptors on noradrenergic terminals in mouse and rat rain cortex

Summary Brain cortex slices were preincubated with ³H-noradrenaline and superfused with physiological salt solution containing desipramine. We studied the inhibition of the electrically evoked tritium overflow caused by histamine in the presence of -adrenoceptor ligands (mouse and rat brain cortex), and the inhibition caused by talipexole (the former B-HT 920) in the presence of H₃-receptor ligands (mouse brain cortex).In mouse brain cortex slices, the inhibitory effect of histamine on the tritium overflow evoked by 36 pulses, 0.3 Hz was not changed by the ₁-adrenoceptor antagonist prazosin, but increased by the ₂-adrenoceptor antagonist rauwolscine. When the current strength or the duration of electrical pulses was reduced to compensate for the increase in evoked tritium overflow produced by rauwolscine, the latter still. enhanced the effect of histamine. The histamine-induced inhibition of tritium overflow evoked by 360 pulses, 3 Hz was not affected by the ₁-adrenoceptor agonist phenylephrine but attenuated by the ₂-adrenoceptor- agonist talipexole. Finally, the inhibition by histamine of the tritium overflow evoked by 3 pulses, 100 Hz was attenuated by talipexole but not affected by rauwolscine. Conversely,. the inhibitory effect of talipexole on tritium overflow elicited by 360 pulses, 3 Hz was slightly attenuated by the H₃-receptor agonist R-(–)--methylhistamine but not, affected by the H₃- receptor antagonist thioperamide. In rat brain cortex slices, histamine only tended to inhibit tritium overflow evoked by 360 pulses, 3 Hz, both in the absence of -adrenoceptor antagonists and in the presence of prazosin. However, histamine markedly inhibited the evoked overflow in the presence of rauwolscine. Again, enhancement of the histamine-induced inhibition also occurred when the current strength or the duration of pulses was reduced in order to compensate for the increase in evoked tritium overflow produced by rauwolscine.The results suggest that the ₂-autoreceptors and the H₃-heteroreceptors at the noradrenergic nerve endings in the brain of mouse and rat interact with each other. Activation of the ₂-autoreceptors decreases, whereas blockade of the activated (but not of the non-activated) ₂-autoreceptors increases, the inhibitory effect of histamine. Activation of the H3-heteroreceptors slightly decreases, whereas blockade of the H₃-receptors fails to affect, the inhibitory effect of talipexole.Send offprint requests to E. Schlicker at the above address     

Presynaptic alpha 2-adrenoceptors modulate the release of [3H]noradrenaline from rat spinal cord dorsal horn neurones

Slices of the dorsal half of the rat spinal cord were used to investigate the existence of a noradrenergic feedback modulation of noradrenaline release. After crude preparation of the vertebral column, the spinal cord was ejected by hydraulic pressure and transverse slices were cut. These were preincubated with [3H]noradrenaline during 0.1 Hz electrical stimulation and then superfused and stimulated electrically for two periods. The stimulation-evoked release of [3H]noradrenaline was Ca2+-dependent and tetrodotoxin-sensitive. Pretreatment of the animals with the noradrenergic neurotoxin, DSP-4, reduced the tritium content in the slices and the stimulation-evoked release to less than 10% of the controls. Clonidine (0.01-1 microM) inhibited the evoked overflow by 60% maximally and yohimbine (0.1-1 microM) enhanced it by 160% maximally. The effects of clonidine were antagonized by yohimbine. These results provide evidence that noradrenaline release from spinal cord slices is controlled by an alpha 2-adrenoceptor-mediated, negative feedback mechanism.