Chronic clonidine induces functional down-regulation of presynaptic alpha 2-adrenoceptors regulating [3H]noradrenaline and [3H]5-hydroxytryptamine release in the rat brain

Clonidine inhibited, through the activation of alpha 2 presynaptic receptors, the release of [3H]noradrenaline (pIC30 = 7.47) and [3H]5-hydroxytryptamine (pIC30 = 6.47) evoked by 15 mM KCl from superfused rat cerebral cortex synaptosomes. The natural agonist noradrenaline inhibited the K+-evoked release of the two [3H]amines less effectively after long-term (12 days) treatment with clonidine than after acute treatment. It can be concluded that chronic clonidine administration can induce down-regulation of both the alpha 2 presynaptic autoreceptors located on noradrenaline terminals and the alpha 2 presynaptic heteroreceptors located on serotonin terminals.     

Acetylcholine release from rat hippocampal slices is modulated by 5-hydroxytryptamine

Experiments were performed with slices of rat hippocampus in order to investigate whether the release of acetylcholine in this area is modulated through 5-hydroxytryptamine (5-HT) receptors. The slices were prelabeled with [3H]choline then stimulated electrically twice for 4 min each at a frequency of 3 Hz. The overflow of tritium evoked was inhibited by exogenous 5-HT in a concentration-dependent manner. The 5-HT2 receptor agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HC1 ((+/-)-DOI), did not mimic 5-HT. The effect of 5-HT was antagonized by methiothepin but not by the 5-HT2 antagonist, ketanserin. The 5-HT1 agonist, 5-methoxy-3-[1,2,3,6-tetrahydropyridin-4-yl]-1H-indole (RU 24969), inhibited the electrically evoked overflow of tritium, whereas the 5-HT1A-selective agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), was ineffective. Methiothepin itself, but not ketanserin, increased the evoked overflow of tritium. In contrast, the overflow was inhibited by the 5-HT uptake blocker, 6-nitroquipazine. The evoked overflow was also reduced by d-fenfluramine, a serotonin releaser. The concentration-inhibition curve for d-fenfluramine was shifted to the right by methiothepin. It is concluded that the release of ACh in rat hippocampus may be tonically inhibited by 5-HT through the activation of receptors, possibly belonging to the 5-HT1B subtype.     

Release of [3H]noradrenaline induced by 5-hydroxytryptamine from cat pial arteries.

Pial arteries of cats were used to analyse the effects of 5-hydroxytryptamine (5-HT) on the release of [3H]noradrenaline. To achieve this the vessels were preincubated with [3H]noradrenaline and the effect of different concentrations of 5-HT (10(-6), 10(-5), 10(-4) M) on the release of tritium was studied. 5-HT elicited release of radioactivity in a dose-dependent manner. Removal of both superior cervical sympathetic ganglia 15 days before the experiment of pretreatment of the animals with reserpine (3 mg kg-1, total dose) produced a significant decrease in the outflow of tritium induced by 5-HT. In these arteries, the amount of radioactivity retained at the end of the experiment was much diminished. Cocaine (10(-6) M) caused a significant decrease in the tritium efflux induced by 5-HT (1"0(-5) M). These results show that 5-HT has an indirect adrenergic effect in the pial arteries of the cat only at high doses of 5-HT, and confirm that sympathetic innervation of these vessels mainly comes from the superior cervical ganglia.     

Nicotine-evoked [3H]5-hydroxytryptamine release from rat striatal synaptosomes

The aim of this study was to characterize the pharmacology of presynaptic nicotinic cholinoceptors (nAChRs) that modulate release of 5-hydroxytryptamine (5-HT) from superfused rat brain synaptosomes preloaded with [3H]5-HT. Nicotine increased 5-HT release from striatal synaptosomes (maximally by 15-30%) but not from cerebral cortex or hippocampal synaptosomes. Release of striatal 5-HT was increased in a concentration-dependent manner by nicotine, epibatidine, cytisine, and ACh (with added esterase inhibitor and muscarinic antagonist). Respective EC50 values were: 0.5, 0.003, 0.1 and 0.7 microM. The maximal effect of each agonist was virtually completely blocked by a high concentration of the insurmountable nicotinic antagonist mecamylamine; at a higher concentration of epibatidine (3 microM), a mecamylamine-insensitive effect was revealed. Nicotine, ACh and epibatidine appeared equally efficacious, whereas cytisine was of lower efficacy (60-70% of ACh). Release evoked by a half-maximal concentration of nicotine was inhibited by the nicotinic antagonists dihydro-beta-erythroidine (IC50 0.04 microM) and methyllycaconitine (IC50 0.06 microM). Nicotine-evoked 5-HT release was not reduced by tetrodotoxin given in a concentration that blocked veratridine-evoked release. These findings provide functional evidence for a direct action of nicotine on 5-HT neurons in the brain. The presynaptic nAChRs that modulate striatal 5-HT release appear to possess a novel pharmacological profile.     

Presynaptic alpha 2-adrenoceptor modulation of 5-hydroxytryptamine and noradrenaline release from vascular adrenergic nerves

Modulation of the stimulation-evoked release of 5-hydroxytryptamine (5-HT) and noradrenaline (NA) by presynaptic alpha 2-adrenoceptors was characterized in the perfused mesenteric vascular bed of the rat. The vasoconstrictor response to periarterial nerve stimulation (PNS; 8 Hz), previously abolished in the presence of 30 nM prazosin, was restored after 15 min treatment with 10 microM 5-HT, without a significant effect on the pressor response to 1 nmol of infused NA, which was previously abolished with prazosin. The restored pressor response to PNS was abolished by 100 nM tetrodotoxin and 100 nM ketanserin. Clonidine (1-10 microM) in the presence of prazosin induced a dose-dependent potentiation of the restored pressor response to PNS after 5-HT treatment while BHT 920 (10 nM-1 microM) and 100 nM clonidine inhibited the restored response. In the presence of 100 nM phentolamine, the restored pressor response to PNS was not altered by clonidine, but was inhibited by BHT 920. The PNS (8 Hz)-evoked tritium release in a preparation labeled with [3H]5-HT was facilitated by clonidine (100 nM-10 microM) while BHT 920 (10 nM-1 microM) and cocaine (1-10 microM) reduced the release. Yohimbine (1 microM) antagonized the effects of clonidine and cocaine but not of BHT 920 on the PNS-evoked tritium release. In the preparation labeled with [3H]NA, clonidine did not alter the PNS-evoked tritium release while BHT 920 inhibited it and cocaine facilitated it. Yohimbine did not antagonize the effect of BHT 920.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potentiation by deprenyl of the autoreceptor-mediated inhibition of [3H]-5-hydroxytryptamine release by 5-methoxytryptamine

Summary The 5-hydroxytryptamine (5HT) receptor agonist, 5-methoxytryptamine, inhibited in a concentration-dependent manner the electrically-evoked release of ³H-5HT from superfused rat hypothalamic slices, with an IC₅₀ of 560 nmol/l, without affecting the spontaneous outflow of radioactivity. In the presence of the selective monoamine oxidase B (MAO B) inhibitor, (–)-deprenyl (1 mol/l), the concentration-effect curve for 5-methoxytryptamine was shifted significantly to the left, and the IC₅₀ was decreased to 25 nmol/l. Under the same experimental conditions, the potency of the 5HT receptor agonist lysergic acid diethylamide (LSD) at inhibiting the electrically-evoked release of ³H-5HT was the same in the presence as well as in the absence of (–)-deprenyl. The IC₅₀ values for LSD were 34 nmol/l in the absence of deprenyl, and 31 nmol/l in the presence of the MAO B inhibitor. It is concluded that deprenyl potentiates the inhibition by 5-methoxytryptamine of ³H-5HT release, by preventing its inactivation through MAO B. Since 5-methoxytryptamine may be present in the pineal gland of some species, the potent effects of this 5-HT receptor agonist on seretoninergic neutrotransmission may be of physiological relevance.     

Presynaptic alpha-adrenoceptors and opiate receptors: inhibition of [3H]noradrenaline release from rat cerebral cortex slices by different mechanisms

The inhibitory effects of morphine and Cd2+ on electrically evoked [3H]noradrenaline release from superfused brain slices were unaffected when release was enhanced by increasing the pulse duration, while the inhibitory effect of noradrenaline and the enhancing effect of phentolamine were diminished. A similar enhancement of [3H]noradrenaline release by 4-aminopyridine reduced the modulatory effects of all drugs examined. Therefore there seem to be different mechanisms for the effect of presynaptic alpha-adrenoceptors and opiate receptors on the availability of Ca2+ for the stimulus-secretion coupling process in noradrenergic nerve terminals.     

Effect of 5-hydroxytryptamine on [3H]-acetylcholine release from guinea-pig striatal slices.

1. The effect of 5-hydroxytryptamine (5-HT) on spontaneous and electrically-evoked tritium efflux was studied in guinea-pig caudate nucleus slices preloaded with [3H]-choline. 2. 5-HT, 10-300 mumol l-1, temporarily increased the spontaneous tritium efflux (as well as the endogenous acetylcholine (ACh) release) and, after 15 min perfusion, inhibited it. The facilitatory effect of 5-HT on spontaneous efflux was increased while the inhibitory effect did not occur in slices taken from dopamine-depleted guinea-pigs. 3. The increase in spontaneous tritium efflux by 5-HT was blocked by methiothepin, methysergide (pA2 8.7) and by the selective 5-HT2 antagonist, ritanserin (pA2 6.7). 4. The inhibition of spontaneous tritium efflux by 5-HT was prevented by methysergide and methiothepin but not by ritanserin and (-)-propranolol. 5. 5-HT, 100 mumol l-1, inhibited the electrically-evoked tritium efflux and this effect was unchanged in dopamine-depleted slices. 6. The inhibition of electrically-evoked tritium efflux by 5-HT was blocked by methiothepin and methysergide but not by (-)-propranolol or ritanserin. 7. These results suggest that 5-HT may exert a rapid and transient (excitatory) and a more prolonged (inhibitory) control over striatal cholinergic neurones.     

Increased cyclic AMP reduces 5-HT1D receptor-mediated inhibition of [3H]5-hydroxytryptamine release from guinea-pig cortical slices

This study investigated the role of cyclic AMP in the inhibition of [³H]5-hydroxytryptamine ([³H]5-HT) release mediated through the 5-HT_1D autoroceptor using slices of guinea-pig cerebral cortex. Forskolin, dibutyryl-cyclic AMP and rolipram, which increase intracellular cyclic AMP by different mechanisms, all attenuated the inhibitory effect of the autoreceptor agonist 5-carboxamidotryptamine. These results indicate that modulation of cyclic AMP production affects 5-HT_1D autoreceptor function.     

The purported dopamine agonist DPI inhibits [3H]noradrenaline release from rat cortical slices but not [3H]dopamine and [14C]acetylcholine release from rat striatal slices in-vitro

The effects of the purported dopamine (DA) receptor agonist (3,4-dihydroxyphenylimino)-2-imidazolidine (DPI) upon the in-vitro K+-induced release of [3H]DA and [14C]acetylcholine from rat neostriatal slices, and of [3H]noradrenaline from rat neocortical slices have been investigated and compared with those of the DA receptor agonist TL-99 and the alpha-adrenoceptor agonist clonidine, respectively. The rapid decomposition of the catechol compounds DPI and TL-99 in the Krebs-Ringer bicarbonate superfusion medium was shown to be inhibited by both the chelating agent EDTA and the reducing agent ascorbic acid. The results suggest that in-vitro DPI is unable to stimulate striatal DA receptors, whereas it is effective in stimulating cortical alpha 2-adrenoceptors (EC50 = 61 nM). It is concluded that DPI should be considered as a mixed alpha 1/alpha 2-adrenoceptor agonist and that the designation of DPI as a DA receptor agonist should be abandoned.     

Stimulation of [3H]norepinephrine release from hippocampal slices by excitatory amino acids

[3H]norepinephrine efflux from preloaded rat hippocampal slices was increased in a dose-dependent manner by excitatory amino acids (EAA) in the following potency order: N-methyl-D-aspartic acid (NMDA) greater than kainic acid greater than L-glutamic acid greater than or equal to D,L-homocysteic acid greater than L-aspartic acid greater than quinolinic acid greater than quisqualic acid. The effect of EAA was blocked by physiological concentration of Mg2+, with the exception of kainic acid. D,L-2-amino-7-phosphonoheptanoic acid (APH) dose-dependently inhibited NMDA effect (IC50 = 69 mumol/L), whereas at 1 mmol/L it was ineffective versus kainic acid. The release of [3H]norepinephrine induced by quinolinic acid was blocked by APH 0.1 mmol/L. gamma-D-glutamylglycine dose-dependently inhibited kainic acid effect with an IC50 = 1.15 mmol/L. Tetrodotoxin 2 mumol/L reduced NMDA and kainic acid effects by 40 and 20%, respectively. The data indicate a possible involvement of central noradrenergic system in the modulation of excitotoxic action of EAA and offer a reliable system for testing new compounds acting at EAA-receptors by measuring norepinephrine release in vitro.     

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.     

Lack of α2-adrenoceptor autoregulation of noradrenaline release in rat nucleus accumbens slices

The role of release-inhibitory α₂-adrenoceptors on or near noradrenergic nerve terminals was investigated in slices of rat nucleus accumbens and medial prefrontal cortex, representing major projection areas of mesocorticolimbic dopamine neurons. As expected, the electrically evoked release of [³H]noradrenaline from superfused medial prefrontal cortex slices was strongly inhibited (to about 25% of control values) by exogeneous noradrenaline and by the α₂-adrenoceptor agonist oxymetazoline, while blockade of α₂-adrenoceptors with phentolamine or rauwolscine caused a large increase (to almost 250% of control values) in neurotransmitter release. In striking contrast and much to our surprise, these drugs did not at all affect the electrically evoked release of [³H]noradrenaline from nucleus accumbens slices. These results indicate that α₂-adrenoceptor autoregulation of noradrenaline release may not be a major feature of noradrenergic varicosities in rat nucleus accumbens. Thus, the functioning or presence of these receptors may vary with the brain region innervated by locus coeruleus neurons and may not represent a ubiquitous feature of noradrenergic neurons. Received: 9 September 1997 / Accepted: 22 September 1997     

Pre-junctional effects of oximes on [3H]-acetylcholine release in rat hippocampal slices during soman intoxication

In this study, the non-reactivating effects of oximes in the hippocampus of the rat are investigated. The potassium (51 mM) evoked release of [(3)H]-acetylcholine and the liberation of [(3)H]-choline were determined in hippocampal slices following in vitro exposure to soman and five oximes (toxogonin, HI-6, HL?-7, P2S and 2-PAM) in separate experiments by superfusion. In the absence of soman, toxogonin and HL?-7 in particular induced a concentration dependent significant increase in the evoked release of [(3)H]-acetylcholine. There was also a significant effect of HI-6, but the effect was much smaller. Two pralidoxime salts, P2S (methanesulfonate salt) and 2-PAM (methiodide salt), had similar but lower effects that were only observed at relatively high concentrations. Experiments performed following complete inhibition of the acetylcholinesterase activity by soman (1.0 microM) showed that HI-6 and HL?-7 induced a significant decrease in the potassium-evoked release of [(3)H]-acetylcholine, while the liberation of [(3)H]-choline increased. Toxogonin, P2S and 2-PAM did not reduce significantly the evoked release of [(3)H]-acetylcholine. Only limited reactivation of the acetylcholinesterase activity was observed in superfusion experiments with toxogonin, HI-6, P2S and 2-PAM following exposure of hippocampal slices to soman. However, HL?-7 was proved to be relatively more effective in reactivating the acetylcholinesterase activity at high concentrations (50 and 200 microM). The acetylcholinesterase activity was reactivated to approximately 12% and 40% of control, respectively. It is concluded that HI-6 and HL?-7 have important non-acetylcholinesterase reactivating properties following soman poisoning, as may be seen by the significant reduction in the evoked release of [(3)H]-acetylcholine effected by these oximes. HL?-7 is of particular interest in view of its ability to additionally improve reactivation of the acetylcholinesterase activity.     

Stimulation by vanadate of [3H]noradrenaline release from rabbit pulmonary artery and its inhibition by noradrenaline

Vanadate, the +5 oxidation state of vanadium, present in mammalian tissues, even in nerve tissue, and a competitive inhibitor of NaK-ATPase, significantly enhanced the release of [3H]noradrenaline evoked from rabbit isolated perfused pulmonary artery by electrical stimulation. Its effect proved to be concentration-dependent. Noradrenaline (10(-6) M) reduced the vanadate-potentiated release of [3H]noradrenaline. The effect of noradrenaline is mediated via alpha 2-adrenoceptors as evidenced by the finding that yohimbine 3 x 10(-7) M prevented its action. The effect of vanadate was dependent on external K ions. When the effect of vanadate on [3H]noradrenaline release was studied under conditions when the NaK-ATPase enzyme activity was inhibited by removal of external K for 45 min, vanadate was ineffective. This finding indicates that the effect is related to the inhibition of NaK-ATPase activity, a condition known to result in transmitter release.     

Pharmacological characterization of presynaptic α-adrenoceptors modulating [3H]noradrenaline and [3H]5-hydroxytryptamine release from slices of the hippocampus of the rat

The experiments served to characterize the receptors mediating the inhibitory effect of α-adrenergic drugs on K⁺ (20 mM)-induced [³H]noradrenaline (NA) and [³H]5-hydroxytryptamine ([³H]5-HT) release from slices of the dorsal part of rat hippocampus. Dose-response curves were constructed using the cumulative dose-response technique (Frankhuyzen and Mulder, 1982). All of the adrenergic agonist drugs examined inhibited the K⁺-induced [³H]NA release. NA appeared to have the highest intrinsic activity followed by adrenaline. Clonidine and adrenaline had similar intrinsic activities, while that of oxymetazoline was lowest. The highest pD₂ values were observed for oxymetazoline and clonidine, being slightly higher than that of adrenaline followed by NA. By far the lowest pD₂ values was observed for phenylephrine. With the exception of phenylephrine, all of the agonists also inhibited the K⁺-induced [³H]5-HT release. NA, adrenaline and oxymetazoline appeared to have similar intrinsic activities, while that of clonidine was considerably lower. The pD₂ values of NA and adrenaline were not significantly different but were somewhat lower than those of oxymetazoline and clonidine. Similar antagonistic effects of phentolamine and yohimbine were observed with respect to the adrenergic inhibition of K⁺-induced [³H]NA and [³H]5-HT release. Prazosin, however, appeared to be ineffective in both instances. It is concluded from these results that the presynaptic adrenergic inhibition of [³H]NA as well as [³H]5-HT release is mediated by α₂-adrenoceptors located on noradrenergic and serotonergic varicosities, respectively. Furthermore, our data suggest that these α₂-adrenoceptors are not pharmacologically identical.     

High affinity binding of [3H]6-nitroquipazine to cortical membranes in the rat: inhibition by 5-hydroxytryptamine and 5-hydroxytryptamine uptake inhibitors

[3H]6-Nitroquipazine is a new, suitable radioligand for studying the uptake system for 5-hydroxytryptamine (5-HT; serotonin). In the present study, inhibition by drugs of the binding of [3H]6-nitroquipazine to uptake sites for 5-HT in the cerebral cortex of the rat was investigated. The inhibition of 5-HT and several inhibitors of the uptake of 5-HT (paroxetine, clomipramine, citalopram, Z-norzimelidine, fluoxetine, imipramine, desipramine and 5-methoxytryptoline) against the binding of [3H]6-nitroquipazine to membranes from the cortex of the rat were the same and competition curves indicated a single population of binding sites. The addition of 5-HT and the tricyclic inhibitors of the uptake of 5-HT, imipramine, clomipramine and desipramine, all produced changes in the apparent dissociation constant (Kd), without changes in the number of binding sites (Bmax). Also, the non-tricyclic inhibitors of the uptake of 5-HT, paroxetine, citalopram, fluoxetine and Z-norzimelidine, and 5-methoxytryptoline, all produced changes in Kd values without changes in the Bmax. These results suggest that all the drugs used in this experiment exhibited competitive interactions with the binding of [3H]6-nitroquipazine to uptake sites for 5-HT in the brain of the rat. These drugs may bind to common binding sites, which are likely to represent the substrate recognition sites for the uptake of 5-HT.