A contribution, from a possible local anaesthetic action, to the effects of yohimbine on evoked noradrenaline overflow

Both yohimbine (0.1 to 10 microM) and phentolamine (10 microM) increased the tritium overflow evoked by electrical stimulation (2.5 Hz, 1 ms, 15 V, for 90 s every 20 min) of mouse isolated vas deferens previously incubated with (-)-[3H]noradrenaline. At their maximally effective concentrations, phentolamine (10 microM) produced an effect that was sustained over the 2 h of the experiment while the effect of yohimbine (6 microM) decreased by about 60% over the first 40 min and was then sustained at a lower level. At higher concentrations of yohimbine, the increase in evoked tritium overflow was less marked and at the highest concentration tested (30 microM) evoked overflow was reduced below the levels seen before exposure to the drug. It is concluded that at concentrations maximally effective in inhibiting the presynaptic alpha-adrenoceptor-mediated mechanism controlling transmitter release, the known local anaesthetic effect of yohimbine may contribute to the overall effect on evoked transmitter overflow.     

Effect of blockade of noradrenaline re-uptake on evoked tritium overflow from mouse vasa deferentia and rat cortex slices.

1. In tissues previously incubated with [3H]-noradrenaline exposure to cocaine (0.1 to 10 microM) or desmethylimipramine (0.01 to 1 microM) produced a concentration-dependent increase (up to 2 fold) in electrically evoked (3 Hz, 2 ms, 20 mA, 120s every 20 min) fractional overflow of tritium from rat brain cortex slices but not from mouse vas deferens (2.5 Hz, 2 ms, 400 mA, for 90s every 14 min). 2. Yohimbine and idazoxan (0.01 to 1 microM) increased fractional evoked overflow of tritium by up to 2 fold; in the presence of these drugs, cocaine (10 microM) produced an increase in both tissues (up to 3.5 fold over control). 3. In brain slice an increase in stimulation frequency (0.1, 0.5, 1, 3 and 6 Hz) decreased fractional evoked overflow of tritium per pulse but cocaine (10 microM) produced a significant enhancement at each frequency except 6 Hz. In vas deferens fractional tritium overflow per pulse changed little with increasing frequency and cocaine produced no effect. 4. In both tissues fractional evoked overflow of tritium was dependent on the stimulation current; cocaine (10 microM) increased fractional evoked overflow from brain slice at every current tested but was without effect in vas deferens. 5. Chromatographic separation of the released tritium showed there was little difference in the proportions of [3H]-noradrenaline and 3H-metabolites overflowing from the tissues. Cocaine increased the proportion of [3H]-noradrenaline and decreased the proportion of [3H]-DOPEG overflowing both at rest and during stimulation. 6.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antagonistic Properties of RU 24969, a Preferential 5-HT1 Receptor Agonist,at Presynaptic α2-Adrenoceptors of Central and Peripheral Neurones

Abstract: The effect of RU 24969 (5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole) on the electrically evoked ³H overflow was studied in superfused rat brain cortex slices preincubated with ³H-noradrenaline or ³H-serotonin and in superfused segments of the rat vena cava preincubated with ³H-noradrenaline. In cortex slices preincubated with ³H-noradrenaline, RU 24969 facilitated the electrically (3 Hz) evoked ³H overflow. This effect was abolished by phentolamine but was not affected by desipramine or the 5-HT₃ receptor antagonist ICS 205–930. The concentration-response curve of noradrenaline for its inhibitory effect on the evoked overflow (determined in the presence of desipramine) was shifted to the right by RU 24969 32 and 100 μmol/1. In this respect, RU 24969 was about 500 times less potent than phentolamine. In cortex slices preincubated with ³H-serotonin, the inhibitory effect of 3.2 μmol/1 RU 24969 on the electrically evoked ³H overflow was increased by phentolamine. In segments of the vena cava, RU 24969 inhibited the electrically (0.66 Hz) evoked ³H overflow. The concentration-response curve of RU 24969 was U-shaped, since at concentrations higher than 0.1 μmol/1 the extent of inhibition decreased with increasing concentrations of RU 24969. In the presence of phentolamine, the concentration-dependent attenuation of the RU 24969-induced inhibition of overflow was no longer detectable. The present results suggest that RU 24969 is a weak antagonist at presynaptic α₂-adrenoceptors (by more than 2.5 log units less potent than as an agonist at presynaptic 5-HT_1B auto- and heteroreceptors).     

Receptor protection experiments confirm the identity of presynaptic α2-autoreceptors

Receptor protection experiments were carried out in cerebrocortical slices from rabbits in order to study the sites at which drugs with alpha-adrenoceptor affinity modulate the release of noradrenaline. The slices were preincubated with 3H-noradrenaline. They were then superfused with 3H-noradrenaline-free medium and stimulated electrically (3 Hz) twice for 2 min each, after 60 and 250 min of superfusion (S1, S2). Phenoxybenzamine was added from 85 to 95 min of superfusion. Potential protecting drugs were present for 5 min before and during the exposure to phenoxybenzamine and then washed out together with the latter. Phenoxybenzamine 0.1 and 1 mumol/l increased the evoked overflow of tritium by 77 and 287%, respectively, as indicated by the S2/S1 overflow ratio. When cocaine was present throughout superfusion, phenoxybenzamine 0.1 and 1 mumol/l increased the evoked overflow by 97 and 353%, respectively. Clonidine 0.1-100 mumol/l, when added before and during the contact with phenoxybenzamine, reduced or even abolished the increase caused by the latter. This interaction was not changed when cocaine was included in the superfusion fluid. The increase caused by phenoxybenzamine was also reduced or abolished by noradrenaline 1-100 mumol/l (tested in the presence of cocaine), yohimbine 0.01-1 mumol/l and phentolamine 0.1-10 mumol/l. Only high concentrations of clonidine, noradrenaline, yohimbine and phentolamine changed the evoked overflow when given alone (and subsequently washed out). The effect of phenoxybenzamine was not modified by prazosin 1 mumol/l, morphine 1 mumol/l and naloxone 10 mumol/l.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endogenous noradrenaline as modulator of hippocampal serotonin (5-HT)-release

Summary The modulation of hippocampal serotonin (5-HT)-release by noradrenaline was studied in rabbit hippocampal slices, which were preincubated with ³H-serotonin and then superfused continuously. Electrical field stimulation of the slices elicited a tritium overflow, which was decreased by clonidine in a concentration dependent manner. Phentolamine antagonized the effects of clonidine and, given alone, increased the evoked tritium overflow. This facilitatory effect of phentolamine was further enhanced in the presence of (+)oxaprotilin, a highly selective noradrenaline uptake inhibitor, whereas the (-)enantiomer of oxaprotilin, which does not affect noradrenaline uptake, was inactive. (+)Oxaprotilin but not (-)oxaprotilin, given alone, inhibited the evoked tritium overflow. The inhibitory effect of (+)oxaprotilin was antagonized by phentolamine.In the presence of phentolamine, the -adrenoceptor antagonists yohimbine, rauwolscine and corynanthine decreased the evoked 5-HT-release concentration dependently. Their inhibitory effects were, however, abolished (corynanthine) or inversed to a facilitation of release (yohimbine, rauwolscine) if instead of phentolamine the 5-HT-receptor antagonist metitepin was present. Therefore we suggest that yohimbine, rauwolscine and corynanthine, in addition to their -adrenoceptor antagonistic properties, may act as agonists at 5-HT-autoreceptors. Possibly the indol part of their molecules is responsible for this effect. Furthermore, our results provide evidence for the modulation of hippocampal 5-HT-release by endogenous noradrenaline.This study was supported by the Deutsche Forschungsgemeinschaft (SFB 70)     

Influence of fentanyl,levorphanol and pethidine on the release of noradrenaline from rat brain cortex slices

Summary In slices of rat brain cortex preincubated with (–)-³H-noradrenaline, the influence of fentanyl, levorphanol and pethidine on the efflux of tritium was investigated. The spontaneous outflow of tritium was not changed by low, and was accelerated by high concentrations of the drugs. The overflow of tritium evoked by electrical stimulation at 3 Hz was diminished by 10^–8–10^–7 M fentanyl and by 10^–7–10^–6 M levorphanol, but was augmented by 10^–5 M levorphanol. Naloxone prevented the inhibitory effect of fentanyl and levorphanol. In contrast to fentanyl and levorphanol, pethidine did not decrease, but at concentrations of 10^–6–10^–5 M greatly increased the stimulation-induced overflow of tritium. However, the increase was abolished, and the stimulation-evoked overflow slightly reduced, after the re-uptake of noradrenaline had been blocked by cocaine. It is concluded that fentanyl, levorphanol and pethidine share with morphine the ability to inhibit the release of transmitter from cerebrocortical noradrenaline neurones evoked by nerve impulses.     

Facilitation of noradrenaline release from rat brain slices by \-adrenoceptors

The present study examined whether stimulation of \-adrenoceptors facilitated noradrenaline release in the rat brain. Electrical stimulation-evoked overflow of tritium from rat cerebral cortical, hypothalamic and hippocampal slices labelled with ³H-noradrenaline was measured during superfusion for 100 min. Tissue slices were electrically simulated (1 Hz, 20 mA, 2 ms, 2 min), at 20(S1) and 70(S2) min after the onset of superfusion. The nonselective \-adrenoceptor agonist isoproterenol (0.1 – 10 nM) enhanced stimulation-evoked overflow of tritium from slices of cerebral cortex, hypothalamus and hippocampus in a concentration-dependent manner; mean S2/S1 ratios with 10 nM isoproterenol were 161 +- 11%, 142 +- 15% and 143 - 12% of control, respectively, in the three brain regions. The facilitatory effect of isoproterenol in cerebral cortical slices was antagonized by propranolol (50 nM), a nonselective \sb-adrenoceptor antagonist, and by the \sb₁- and \sb₂-selective adrenoceptor antagonists ICI 89,406 (1 nM) and ICI 118,551(1 nM), respectively. The \sb₁- and \sb₂-selective adrenoceptor agonists prenalterol and albuterol (0.1 \2- 10 nM), respectively, also increased stimulation-evoked overflow of tritium from cerebral cortical slices; these effects were antagonized by \sb-adrenoceptor antagonists. These findings suggest that stimulation of \sb-adrenoceptors enhance noradrenaline release from rat cerebral cortical, hypothalamic and hippocampal slices; this release mechanism appears to involve both \sb₁- and \sb₂-adrenoceptor subtypes. These facilitating presynaptic receptors may be involved in mediating the antidepressant-like behavioral effects of \sb₂-adrenoceptor agonists.     

Phenoxybenzamine blocks dopamine autoreceptors irreversibly: Implications for multiple dopamine receptor hypotheses

Phenoxybenzamine in μM concentrations increased the electrically evoked overflow of recently taken up [³H]dopamine from superfused slices of cat caudate nucleus, an effect which is also observed for dopamine receptor antagonists. The magnitude of the increase in electrically evoked [³H]dopamine release caused by 1 μM phenoxybenzamine was equal to that elicited by maximally effective concentrations of the specific dopamine receptor antagonist, S-sulpiride. Phenoxybenzamine (1 μM) completely antagonized the inhibition of [³H]dopamine release caused by the dopamine receptor agonist pergolide (10 nM). The α-adrenoceptor antagonist phentolamine (1 μM) had no effect on the electrically evoked overflow of [³H]dopamine, ruling out the possibility that the effect of phenoxybenzamine could be attributed to α-adrenoceptor blockade. A 20 min exposure to 1 μM phenoxybenzamine increased the electrically evoked [³H]dopamine overflow even after the tissue had been washed for two and a half hours. When the caudate slices were exposed for 30 min to the reversible dopamine receptor antagonist S-sulpiride (1 μM) and washed for two and a half hours, no similar increase in stimulation-evoked [³H]dopamine overflow was observed. When sulpiride (1 μM) was present during the exposure to phenoxybenzamine (1 μM), no increase in electrically evoked [³H]dopamine overflow was observed after the washout period. Thus phenoxybenzamine at 1 μM appears to block irreversibly the dopamine autoreceptor in the caudate nucleus. Phenoxybenzamine has been previously reported to block irreversibly dopamine-stimulated adenylate cyclase (D₁) and neuroleptic receptor binding (D₂). The present demonstration that phenoxybenzamine also blocks the dopamine autoreceptor irreversibly thus supports the view that all currently well-established dopamine receptors are sensitive to phenoxybenzamine.     

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.     

Nociceptin inhibits noradrenaline release in the mouse brain cortex via presynaptic ORL1 receptors

A fourth type of opioid receptor, termed ORL₁, has been cloned and nociceptin (also known as orphanin FQ) has been identified as an endogenous ligand at this receptor. We examined whether nociceptin affects the release of noradrenaline in the brain. For this purpose, cerebral cortex slices from the mouse, rat or guinea-pig were preincubated with [³H]noradrenaline and then superfused with medium containing desipramine and rauwolscine. Tritium overflow was evoked electrically (0.3 Hz) or by introduction of Ca²⁺ 1.3 mM into Ca²⁺-free K⁺-rich (15 mM) medium. Nociceptin 1 μM reduced the electrically evoked tritium overflow from mouse, rat and guinea-pig brain cortex slices by 80, 71 and 36%, respectively. Naloxone 10 μM did not change the effect of nociceptin. All subsequent experiments were performed on mouse brain cortex slices and in the presence of naloxone 10 μM. The concentration-response curve of nociceptin (maximum inhibition by 80%, pEC₅₀ 7.5) was shifted to the right by the non-selective ORL₁ receptor antagonist naloxone benzoylhydrazone and the selective ORL₁ receptor antagonist [Phe¹ψ(CH₂-NH)Gly²]-nociceptin(1–13)NH₂ (pA₂ 6.6 and 7.2, respectively). Naloxone benzoylhydrazone did not affect the evoked overflow by itself whereas [Phe¹ψ(CH₂-NH)Gly²]-nociceptin(1–13)NH₂ caused an inhibition by maximally 35% (pEC₅₀ 7.0; intrinsic activity α 0.45). The inhibitory effect of [Phe¹ψ(CH₂-NH)Gly²]-nociceptin(1–13)NH₂ was counteracted by naloxone benzoylhydrazone. Nociceptin also reduced the Ca ²⁺ -evoked tritium overflow in mouse brain cortex slices superfused in the presence of tetrodotoxin. This effect was also antagonized by naloxone benzoylhydrazone, which, by itself, did not affect the evoked tritium overflow. In conclusion, nociceptin inhibits noradrenaline release more markedly in the mouse than in the rat or guinea-pig brain cortex. The effect of nociceptin in the mouse brain cortex involves ORL₁ receptors, which are located presynaptically on noradrenergic neurones. Received: 19 June 1998 / Accepted: 17 July 1998     

Influence of morphine and naloxone on the release of noradrenaline from rat cerebellar cortex slices

Summary In slices of rat cerebellar cortex preincubated with (-)-³H-noradrenaline, the influence of morphine and naloxone on the efflux of tritium was investigated. The spontaneous outflow was not changed by 10^–5 M of either morphine or naloxone. On the other hand, morphine caused a concentration-dependent decrease of the overflow, of tritium evoked by electrical field stimulation. Naloxone did not change the stimulation-induced overflow, but prevented its inhibition by morphine. It is concluded that morphine, through an action on opiate receptors located on cerebellar noradrenergic neurones, inhibits the secretion of the transmitter in response to nerve impulses.     

EFFECTS OF PRAZOSIN,PHENTOLAMINE AND YOHIMBINE ON NORADRENERGIC TRANSMISSION IN THE RAT RIGHT VENTRICLE IN VITRO

1 The effects of prazosin, phentolamine and yohimbine on the accumulation of radioactivity from [³H]-noradrenaline, and on the subsequent spontaneous and field stimulation-evoked outflow of radioactivity have been investigated in the rat right ventricle in vitro. In addition the effects of these agents on contractions produced by exogenous amines or by field stimulation are reported. 2 Prazosin, phentolamine and yohimbine had no effect on the accumulation of radioactivity from [³H]-noradrenaline. 3 The decline in the spontaneous outflow of radioactivity, following loading of the tissue with [³H]-noradrenaline, was reduced by prazosin (1 times 10^-8 or 1 times 10^-7M) and 1 times 10^-6M yohimbine. Phentolamine and 1 times 10^-7M yohimbine had no effect on the spontaneous outflow of radioactivity. 4 Prazosin, phentolamine and yohimbine (all at 1 times 10^-7M) reduced the decline in outflow of radioactivity evoked by field stimulation at 5Hz. In the presence of yohimbine, prazosin or phentolamine had no effect on the evoked outflow. The effect of prazosin, phentolamine and yohimbine alone probably represents antagonism at prejunctional α-adrenoreceptors. 5 The rate of beating and force of contractions evoked by 1 times 10^-6M isoprenaline were not altered by prazosin, phentolamine or 1 times 10^-7M yohimbine. The rate of beating evoked by noradrenaline (1 times 10^-6M) was reduced by prazosin and phentolamine. Yohimbine (1 times 10^-6M) reduced the rate of beating to isoprenaline and to noradrenaline. These effects of prazosin and phentolamine probably represent antagonism at postjunctional α₁-adrenoreceptors and that of yohimbine a decrease in the excitability of the postjunctional membrane. 6 In the control tissues the force of contractions evoked by field stimulation at 5Hz declined slowly with successive stimulations at 30 min intervals. In the presence of prazosin (1 times 10^-8 and 1 times 10^-7M), phentolamine (1 times 10^-7 and 1 times 10^-6M) and 1 times 10^-7M yohimbine the responses did not decline with successive stimulations. Yohimbine (1 times 10^-6M) had no effect on the stimulation-evoked responses. 7 Phentolamine or yohimbine (both at 1 times 10^-7M) had no additional effect on the stimulation-evoked responses in the presence of prazosin (1 times 10^-7M). Prazosin (1 times 10^-8M) and yohimbine (1 times 10^-7M) had no further effect in the presence of phentolamine (1 times 10^-6M). Prazosin or phentolamine (both at 1 times 10^-7M) had no effect on stimulation-evoked responses in the presence of yohimbine (1 times 10^-7M). 8 In the rat right ventricle, the ability of phentolamine and yohimbine to prevent the decline in the stimulation-evoked responses with successive stimulations is probably due to antagonism at prejunctional α-adrenoreceptors. However it is suggested that the ability of prazosin to prevent the decline in response is due to a release of endogenous noradrenaline and to antagonism at prejunctional α-adrenoreceptors.     

Influence of morphine and naloxone on the release of noradrenaline from rat brain cortex slices

Summary In slices of rat brain cortex preincubated with (–)-³H-noradrenaline, the influence of morphine and naloxone on the efflux of tritium was investigated. The spontaneous outflow of tritium was not changed by 10^–7–10^–5 M morphine and by 10^–6–10^–4 M naloxone, but was accelerated by 10^–4 M morphine. Electrical field stimulation augmented tritium outflow. The overflow evoked per ppulse decreased as the frequency of stimulation was increased from 0.3 to 3 Hz, but remained approximately constant when it was further increased to 10 Hz. At frequencies of 0.3, 1, and 3 Hz, but not at 10 Hz, morphine in concentrations of 10^–7–10^–5 M depressed the stimulation-induced overflow of tritium. 10^–4 M morphine did not influence the overflow induced by stimulation at 0.3 and 1 Hz and increased that evoked by stimulation at 10 Hz. Naloxone (10^–6–10^–4 M) did not change the response to stimulation. In the presence of 10^–4 M naloxone, 10^–6 M morphine did not diminish, and 10^–5 M morphine even enhanced the stimulation-induced overflow of tritium. The inhibitory effect of 10^–6 M morphine was not reduced, after tyrosine hydroxylase had been blocked by -methyltyrosine-methylester. It is concluded that morphine through an action on specific opiate receptors inhibits the release of transmitter from cerebrocortical noradrenergic neurones evoked by nerve impulses. By an action unrelated to opiate receptors, morphine at high concentrations increases the stimulation-induced overflow of noradrenaline, presumably by inhibiting its re-uptake into nerve endings.     

H2 receptor-mediated facilitation and H3 receptor-mediated inhibition of noradrenaline release in the guinea-pig brain

The effect of histamine and related drugs on the tritium overflow evoked electrically (0.3 Hz) or by introduction of Ca²⁺ ions into Ca²⁺-free K⁺-rich (25 mmol/l) medium containing tetrodotoxin was studied in superfused guinea-pig brain cortex, cerebellum, hippocampus or hypothalamus slices and in mouse brain cortex slices preincubated with ³H-noradrenaline. The electrically evoked tritium overflow in guinea-pig cortex slices was inhibited by histamine; the H₃ receptor antagonist clobenpropit reversed the effect of histamine to a slight facilitation. The facilitatory effect of histamine (obtained in the presence of clobenpropit) was not affected by the H₁ receptor antagonist mepyramine but abolished by the H₂ receptor antagonist ranitidine. In the absence of clobenpropit, ranitidine augmented the inhibitory effect of histamine. In slices superfused in the presence of ranitidine, the evoked overflow was inhibited by histamine and, more potently, by the H₃ receptor agonist R-α-methylhistamine in a concentration-dependent manner (maximum inhibitory effect obtained for both agonists 30–35%). The concentration-response curve of histamine was shifted to the right by the H₃ receptor antagonist thioperamide. R-α-Methylhistamine inhibited the electrically evoked tritium overflow also in guinea-pig cerebellar, hippocampal and hypothalamic slices. In cortex slices superfused in the presence of clobenpropit, the H₂ receptor agonists impromidine and, less potently, R-sopromidine facilitated the evoked overflow in a concentration-dependent manner. S-Sopromidine only tended to increase the evoked overflow. The effect of impromidine was counteracted by the H₂ receptor antagonists ranitidine and cimetidine. The extent of the maximum facilitatory effect of impromidine (by 15–20%) was about the same when (i) the Ca²⁺ concentration in the medium was reduced from 1.3 to 0.98 mmol/l, (ii) the time of exposure to impromidine was reduced from 28 to 8 min or (iii) cerebellar, hippocampal or hypothalamic slices were used instead of cortical slices. The Ca²⁺-induced tritium overflow in guinea-pig cortex slices was inhibited by histamine (in the presence of ranitidine); this effect was abolished by clobenpropit. In slices superfused in the presence of clobenpropit, impromidine failed to facilitate the Ca²⁺-evoked tritium overflow. The electrically evoked tritium overflow in mouse brain cortex slices was inhibited by histamine by about 60% (both in the absence or presence of ranitidine). The inhibitory effect of histamine was abolished (but not reversed) by clobenpropit. In conclusion, noradrenaline release in the guinea-pig brain cortex is inhibited via presynaptic H₃ receptors and facilitated via H₂ receptors not located presynaptically. In the mouse brain cortex, only inhibitory H₃ receptors occur. The extent of the H₃ receptor-mediated effect is more marked in the mouse than in the guinea-pig brain cortex. Received: 25 September 1997 / Accepted: 17 November 1997     

Cannabinoid CB1 receptor-mediated inhibition of noradrenaline release in the human and guinea-pig hippocampus

We examined the question of whether cannabinoid receptors modulating noradrenaline release are detectable in the brain of humans and experimental animals. For this purpose, hippocampal slices from humans, guinea-pigs, rats and mice and cerebellar, cerebrocortical and hypothalamic slices from guinea-pigs were incubated with [³H]noradrenaline and then superfused. Tritium overflow was evoked either electrically (0.3 or 1Hz) or by introduction of Ca²⁺ ions (1.3μM) into Ca²⁺-free, K⁺-rich medium (25μM) containing tetrodotoxin 1μM. Furthermore, the cAMP accumulation stimulated by forskolin 10μM was determined in guinea-pig hippocampal membranes. We used the following drugs: the cannabinoid receptor agonists (–)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP-55,940) and R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone (WIN 55,212-2), the inactive S(–)-enantiomer of the latter (WIN 55,212-3) and the CB₁ receptor antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide (SR 141716). The electrically evoked tritium overflow from guinea-pig hippocampal slices was reduced by WIN 55,212-2 (pIC_30% 6.5) but not affected by WIN 55,212-3 up to 10μM. The concentration-response curve of WIN 55,212-2 was shifted to the right by SR 141716 (0.032μM) (apparent pA₂ 8.2), which by itself did not affect the evoked overflow. WIN 55,212-2 1μM also inhibited the Ca²⁺-evoked tritium overflow in guinea-pig hippocampal slices and the electrically evoked overflow in guinea-pig cerebellar, cerebrocortical and hypothalamic slices as well as in human hippocampal slices but not in rat and mouse hippocampal slices. SR 141716 (0.32μM) markedly attenuated the WIN 55,212-2-induced inhibition in guinea-pig and human brain slices. SR 141716 0.32μM by itself increased the electrically evoked tritium overflow in guinea-pig hippocampal slices but failed to do so in slices from the other brain regions of the guinea-pig and in human hippocampal slices. The cAMP accumulation stimulated by forskolin was reduced by CP-55,940 and WIN 55,212-2. The concentration-response curve of CP 55,940 was shifted to the right by SR 141716 (0.1μM; apparent pA₂ 8.3), which by itself did not affect cAMP accumulation. In conclusion, cannabinoid receptors of the CB₁ subtype occur in the human hippocampus, where they may contribute to the psychotropic effects of cannabis, and in the guinea-pig hippocampus, cerebellum, cerebral cortex and hypothalamus. The CB₁ receptor in the guinea-pig hippocampus is located presynaptically, is activated by endogenous cannabinoids and may be negatively coupled to adenylyl cyclase. Received: 5 June 1997 / Accepted: 6 August 1997     

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     

Presynaptic serotonin receptors regulate the release of 3H-serotonin in hypothalamic slices of the rabbit

Summary Hypothalamic slices of the rabbit brain were incubated with 10^–7 M of ³H-serotonin (³H-5HT). After the incubation and an initial washout period, a nearly constant basal efflux of tritium was detected. This basal efflux was not significantly altered by Ca²⁺-free solution or by the 5HT-antagonist metitepin (10^–5 M), but was augmented by chlorimipramine (10^–5 M) and by unlabelled 5HT (10^–6 M); the acceleration caused by unlabelled 5HT was absent in presence of chlorimipramine (10^–5 M). Both electrical stimulation (4 Hz, 50 mA, 2 min) and high K⁺ (50 mM) induced an overflow of ³H. This overflow was nearly abolished in Ca²⁺-free solution. In presence of chlorimipramine (10^–5 M) both the tritium overflow evoked by electrical stimulation and that evoked by high K⁺ were augmented by metitepin (10^–5 M) and decreased in a concentration dependent manner by unlabelled serotonin (10^–8–10^–6 M); the latter effect was antagonized by metitepin (10^–6 M and 10^–5 M). These experiments suggest that in rabbit hypothalamic slices, the release of ³H-5HT is controlled by a negative feedback mechanism acting via presynaptic serotonin receptors.     

Inhibition of noradrenaline release in the rat brain cortex via presynaptic H3 receptors

Summary The effects of histamine and related drugs on the evoked tritium overflow from superfused rat brain cortex slices preincubated with³H-noradrenaline were determined. Tritium overflow was stimulated electrically (3 Hz; slices superfused with normal physiological salt solution) or by introduction of CaCl₂ 1.3 mmol/l (slices superfused with Ca²⁺-free medium containing K⁺ 20 mmol/l).Histamine slightly decreased the electrically evoked^H overflow in slices superfused in the presence of desipramine. The degree of inhibition obtained with histamine was doubled when both desipramine and phentolamine were present in the superfusion medium (pIC₁₅ 6.46). Under the latter condition, the evoked overflow was inhibited by the H₃ receptor agonist R-(–)--methylhistamine and its S-(+) enantiomer (pIC₁₅ 7.36 and 5.09, respectively), but was not affected by the H₂ receptor agonist dimaprit and the H₁ receptoragonist 2-thiazolylethylamine (both at up to 32 µmol/l). The concentration-response curve of histamine was shifted to the right by the H3 receptor antagonists thioperamide, impromidine and burimamide (apparent pA₂ 8.37, 6.86 and 7.05, respectively), by the H₂ receptor antagonist ranitidine (apparent pA₂ 4.27) and was not affected by the H₁ receptor antagonist dimetindene (32 µmol/l). The inhibitory effect of R-(–)--methylhistamine on the evoked overflow was also counteracted by thioperamide. Given alone, none of the five histamine receptor antagonists affected the evoked overflow. In the absence of desipramine plus phentolamine, impromidine and burimamide facilitated the electrically evoked³H overflow whereas thioperamide had no effect. The facilitatory effects of impromidine and burimamide were abolished by phentolamine, but not affected by desipramine. The concentration-response curve of noradrenaline for its inhibitory effect on the evoked overflow was shifted to the right by impromidine and burimamide, but not influenced by thioperamide (apparent pA₂ 5.24, 5.04 and <6.5, respectively; experiments carried out in the presence of desipramine). In slices superfused with Ca²⁺-free K⁺-rich medium containing tetrodotoxin, desipramine plus phentolamine, the tritium overflow evoked by introduction of Ca²⁺ was inhibited by histamine; the concentration-response curve of histamine was shifted to the right by thioperamide.The present study shows that the inhibitory effect of histamine on noradrenaline release in the rat brain cortex involves presynaptic H₃ receptors and that the degree of inhibition is increased in the presence of phentolamine. The H₃ receptor antagonists impromidine and burimamide are weak ₂-adrenoceptor antagonists. Send offprint requests to E. Schlicker at the above address     

Pharmacological characterization of the inhibitory effects of neurotensin on the rabbit ileum myenteric plexus preparation.

1 [³H]-amezinium is taken up selectively into noradrenergic axons and their transmitter-storing vesicles and is released from these axons by action potentials. We used it as a non-α-adrenergic marker in order to study the α-adrenergic autoinhibition of noradrenaline release.

2 Rat occipitocortical slices were preincubated with [³H]-amezinium 0.03 μM and then superfused and stimulated electrically (3 Hz for 3 min). The stimulation-evoked overflow of tritium was measured in six groups of slices: from saline-pretreated rats; from saline-pretreated rats, the slices being exposed to exogenous noradrenaline before preincubation with [³H]-amezinium; from saline-treated rats, slices from which were exposed simultaneously to noradrenaline and cocaine before preincubation with [³H]-amezinium; from rats in which noradrenaline stores had been depleted by pretreatment with α-methyltyrosine (α-MT); from α-MT-treated rats, the slices being exposed to noradrenaline before preincubation with [³H]-amezinium; and from α-MT-treated rats, slices from which were exposed to noradrenaline plus cocaine before preincubation with [³H]-amezinium.

3 The stimulation-evoked overflow of tritium, expressed as a percentage of the tritium content of the tissue, was 1.15% in slices from saline-pretreated rats, and was similar in slices from saline-pretreated rats after exposure to noradrenaline or noradrenaline plus cocaine. It was 2.56% in slices from α-MT-treated rats, 1.20% from α-MT-treated rats after exposure to noradrenaline, and 2.88% from α-MT-treated rats after exposure to noradrenaline plus cocaine.

4 Yohimbine 0.1 and 1 μM increased the stimulation-evoked overflow of tritium in slices from all groups of saline-pretreated rats and in those slices from α-MT rats that had been in contact with exogenous noradrenaline. Yohimbine did not change the evoked overflow in slices from α-MT rats that had not been exposed to noradrenaline, or had been exposed to noradrenaline plus cocaine.

5 Clonidine 0.01-1 μM decreased the stimulation-evoked overflow of tritium moderately in slices from saline-pretreated rats, markedly in slices from α-MT-treated rats, and moderately again when the latter slices had been exposed to noradrenaline.

6 It is concluded that the action potential-evoked release of [³H]-amezinium as well as the modulation of this release by yohimbine and clonidine depend on the presence or absence of α-adrenergic autoinhibition caused by the co-secretion of noradrenaline. When there is co-secretion of noradrenaline, the evoked release of [³H]-amezinium is relatively small, yohimbine increases the release, and clonidine can cause only moderate inhibition. When there is no or very little co-secretion of noradrenaline, the evoked release of [³H]-amezinium is at least doubled, yohimbine causes no further increase and clonidine produces strong inhibition.