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The stimulation frequency-noradrenaline release relationship was studied in the vas deferens and the cerebral cortex of NMRI mice, mice in which the alpha2A-, the alpha2B-, the alpha2C- or both the alphaCA- and the alpha2C-adrenoceptor gene had been disrupted (alpha2AKO, alpha2BKO, alpha2CKO and alpha2ACKO), and the wildtype mice from which the knockout animals had been generated. Tissue pieces were preincubated with 3H-noradrenaline and then superfused and stimulated electrically with a constant number of pulses (30 in vas deferens and 50 in brain cortex) at frequencies between 0.03 and 100 Hz. The frequency-evoked tritium overflow curves ascended monophasically in the vas deferens of wildtype and NMRI mice. Disruption of the alpha2B-adrenoceptor gene caused no change. In the vas deferens of alpha2CKO mice, the overflow evoked by low frequencies (0.3 and 1 Hz) was slightly increased. In the vas deferens of alpha2AKO and alpha2ACKO mice, the evoked overflow was increased to a greater extent. Rauwolscine (1 microM) caused a marked increase of the evoked overflow of tritium from the vas deferens of NMRI, wildtype, alpha2BKO and alpha2CKO mice. Rauwolscine also increased the evoked overflow of tritium from the vas deferens of alpha2AKO and alphaC2ACKO mice, but to a smaller extent. The gene disruptions and rauwolscine slightly steepened the slope of the vas deferens frequency-overflow curve. In the brain cortex of wildtype and NMRI mice, the frequency-evoked tritium overflow curves were U-shaped. In the brain cortex of alpha2BKO and alpha2CKO mice, the evoked overflow was slightly reduced. In the brain cortex of alpha2AKO and alpha2AcKO mice, in contrast, the evoked overflow was increased. Rauwolscine (1 microM) caused a marked increase of the evoked overflow of tritium from the brain cortex of NMRI, wildtype, Q2BKO and alpha2CKO mice. Rauwolscine also increased the evoked overflow of tritium from the brain cortex of alpha2AKO and alpha2ACKO mice, but to a smaller extent. The gene disruptions and rauwolscine flattened the U shape of the brain cortex frequency-overflow curve. It is concluded that alpha2-autoinhibition is one factor that shapes the frequency-noradrenaline release relationships in the mouse vas deferens and cerebral cortex. The autoreceptors are mainly alpha2A and to a minor extent, and well detectable in the vas deferens only, alpha2C. When both the alpha2A- and the alpha2C-adrenoceptor have been deleted, alpha2B-adrenoceptors may be expressed as autoreceptors in noradrenergic neurons. It seems possible that alpha2C-autoreceptors depress mainly release at low (around 1 Hz) whereas alpha2A-autoreceptors depress mainly release at high (around 10 Hz) frequencies.

This study investigated the effects of changing the extracellular Ca2+ concentration on facilitation of excitatory junction potential (EJP) amplitude during trains of 20 stimuli at 1 Hz at sympathetic neuroeffector junctions in the guinea-pig vas deferens. These effects were compared with those of Ca2+ channel blockers and agents which act at prejunctional receptors to increase or decrease neurotransmitter release. In these experiments, alpha-adrenoceptor-mediated autoinhibition of neurotransmitter release was blocked by the alpha-adrenoceptor antagonist, phentolamine (1 microM). Varying the extracellular Ca2+ concentration (0.75-6 mM) changed the amplitude of EJPs without affecting the maximum level of facilitation during the trains of stimuli. Reductions in Ca2+ concentration (from 2 mM) were associated with a slowing in the rate of development of facilitation. The Ca2+ channel antagonists, Cd2+ (2 microM and 5 microM) and omega-conotoxin GVIA (10 nM), and agents which act at prejunctional receptors to reduce neurotransmitter release, adenosine (100 microM and 1,000 microM) and prostaglandin E2 (PGE2; 0.1 nM and 1 nM), produced similar effects to those of lowering the extracellular Ca2+ concentration. Raising the extracellular Ca2+ concentration (from 2 mM) increased the rate of development of facilitation. Angiotensin II (AII; 0.5 microM) produced similar effects to raising extracellular Ca2+. However, isoprenaline (1 microM), while increasing EJP amplitude, reduced the maximum level of facilitation and was without effect on the rate of development of facilitation. In the guinea-pig vas deferens EJPs are produced by neurally released ATP. Thus, the findings support the idea that adenosine, PGE2 and AII change ATP release by modifying Ca2+ entry into the nerve terminal. However, the effects of isoprenaline may not solely be accounted for by modifications in Ca2+ entry.

Adaptive phenomena such as desensitization of autoreceptors are considered an important factor in the achievement of therapeutic efficacy of antidepressant drugs after chronic treatment. We have studied whether a chronic treatment with desipramine had a greater effect than a single dose on the extracellular concentrations of noradrenaline in the dorsal hippocampus. Administration of 10 mg/kg i.p. desipramine once daily for 14 days significantly raised the basal extracellular noradrenaline in the dorsal hippocampus 24 h but not 48 h after the last drug injection. A challenge dose of desipramine increased extracellular noradrenaline in rats treated chronically with vehicle and desipramine. The effect was significantly higher in rats treated chronically with desipramine 48 h but not 24 h after the last injection. An intraperitoneal administration of the alpha2-adrenoceptor agonist clonidine at the dose of 10 microg/kg significantly reduced extracellular noradrenaline in the control group but not in animals chronically treated with desipramine whereas 30 microg/kg clonidine produced a similar decrease in both groups. Three concentrations of clonidine (0.05, 0.5 and 1 microM) infused into the hippocampus significantly reduced extracellular noradrenaline to a similar extent in rats chronically treated with saline or desipramine. Fourty-eight hours after the last injection of the chronic treatment, [3H]RX-821002 binding to alpha2-adrenoceptors in the rat locus coeruleus measured by autoradiography was not significantly modified. A slight (17%) but significant decrease of neuronal uptake of [3H]noradrenaline was found in synaptosome preparations from dorsal hippocampus of rats chronically treated with desipramine, but this was likely due to a decrease in affinity. The results suggest that a repeated treatment with desipramine (10 mg/kg i.p. once daily for 14 days) facilitates its effect on extracellular noradrenaline in the dorsal hippocampus and induces adaptive changes probably involving desensitization of alpha2-adrenoceptors, with no changes in their density, on noradrenergic neurons in the locus coeruleus.

We studied whether serotonin release in the CNS is inhibited via cannabinoid receptors. In mouse brain cortex slices preincubated with [3H]serotonin and superfused with medium containing indalpine and metitepine, tritium overflow was evoked either electrically (3 Hz) or by introduction of Ca2+ (1.3 mM) into Ca2+-free K+-rich (25 mM) medium containing tetrodotoxin. The effects of cannabinoid receptor ligands on the electrically evoked tritium overflow from mouse brain cortex slices preincubated with [3H]choline and on the binding of [3H]WIN 55,212-2 and [35S]GTPgammaS to mouse brain cortex membranes were examined as well. In superfused mouse cortex membranes preincubated with [3H]serotonin, the electrically evoked tritium overflow was inhibited by the cannabinoid receptor agonist WIN 55,212-2 (maximum effect of 20%, obtained at 1 microM; pEC50=7.11) and this effect was counteracted by the CB1 receptor antagonist SR 141716 (apparent pA2=8.02), which did not affect the evoked tritium overflow by itself. The effect of WIN 55,212-2 was not shared by its enantiomer WIN 55,212-3 but was mimicked by another cannabinoid receptor agonist, CP-55,940. WIN 55,212-2 also inhibited the Ca2+-evoked tritium overflow and this effect was antagonized by SR 141716. Concentrations of histamine, prostaglandin E2 and neuropeptide Y, causing the maximum effect at their respective receptors, inhibited the electrically evoked tritium overflow by 33, 69 and 73%, respectively. WIN 55,212-2 (1 microM) inhibited the electrically evoked tritium overflow from mouse brain cortex slices preincubated with [3H]choline by 49%. [3H]WIN 55,212-2 binding to mouse cortex membranes was inhibited by CP-55,940, SR 141716 and WIN 55,212-2 (pKi=9.30, 8.70 and 8.19, respectively) but not by the auxiliary drugs indalpine, metitepine and tetrodotoxin (pKi<4.5). [35S]GTPgammaS binding was increased by WIN 55,212-2 (maximum effect of 80%, pEC50=6.94) but not affected by WIN 55,212-3. In conclusion, serotonin release in the mouse brain cortex is inhibited via CB1 receptors, which may be located presynaptically and are not activated by endogenous cannabinoids. The extent of inhibition is smaller than that obtained (1) via another three presynaptic receptors on serotoninergic neurones and (2) via CB1 receptors on cholinergic neurones in the same tissue.

This study was designed to assess the effects of imidazoline drugs on putative presynaptic imidazoline receptors modulating brain monoamine synthesis in vivo. The accumulation of 3,4-dihydroxyphenylalanine (dopa) and 5-hydroxytryptophan (5-HTP) after decarboxylase inhibition was used as a measure of the rate of tyrosine and tryptophan hydroxylation in various brain regions of naive rats and after irreversible alpha2-adrenoceptor inactivation with EEDQ (1.6 mg/kg, i.p., 6 h). Clonidine (1-3 mg/kg), moxonidine (1-10 mg/kg) and rilmenidine (10 mg/kg) (mixed I1/alpha2 agonists) decreased dopa and 5-HTP synthesis in the cerebral cortex (14%-81%), hippocampus (27%-84%) and/or striatum (29%-56%), but these inhibitory effects were abolished in N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ)-treated rats. Similarly, the stimulatory effect of efaroxan (mixed I1/alpha2 antagonist; 10 mg/kg) on dopa synthesis in the cortex (77%) and hippocampus (57%) was abolished by EEDQ. The selective I1-ligand 2-endo-amino-3-exoisopropylbicyclo-heptane (AGN-192403; 5-10 mg/kg) did not modify dopa or 5-HTP synthesis in any brain region in naive or EEDQ-treated rats. Idazoxan (mixed I2/alpha2 antagonist; 20 mg/kg) increased dopa synthesis in the cortex (111%) and hippocampus (87%), but the stimulatory effects were abolished by EEDQ. Moreover, idazoxan and efaroxan decreased 5-HTP synthesis in the cortex (12%-34%) and hippocampus (30%-34%) in a manner sensitive to blockade by the 5-HT1A receptor antagonist WAY 100135. The selective I2-igands 2-(2-benzofuranyl)-2-imidazoline (2-BFI; 20 mg/kg) and 2-styryl-2-imidazoline (LSL 61122; 10 mg/kg) did not alter the synthesis of dopa or 5-HTP in the cortex or hippocampus. In striatum, 2-BFI (1-20 mg/kg) dose-dependently decreased dopa synthesis (ED50: 5.9 mg/kg), reduced dopamine levels (6%-36%) and increased those of its metabolites DOPAC (15%-95%) and HVA (24%-74%). The inhibitory effect of 2-BFI on dopa/dopamine synthesis in striatum remained unchanged after alkylation of imidazoline receptors with isothiocyanatobenzyl imidazoline (IBI; 60 mg/kg, 6 h) or blockade of these receptors with 2-(2-ethyl 2,3-dihydro-2-benzofuranyl)-2-imidazole (KU-14R; 7-20 mg/kg). Therefore, most imidazoline drugs modulated the synthesis of brain monoamines through interaction with alpha2-adrenoceptors or 5-HT1A receptors. The results do not provide functional evidence for the existence of presynaptic imidazoline receptors regulating the synthesis of monoamines in the rat brain.

The existence of presynaptic alpha 2-autoreceptors, and the subtype to which the receptors belong, were studied in the brain cortex from two African green monkeys (Cercopithecus aethiops). Slices of the brain cortex were preincubated with 3H-noradrenaline. The slices were then superfused with medium containing desipramine (1 microM) and stimulated electrically with trains of 72 pulses. 3 Hz. Concentration-response curves of nine alpha-adrenoceptor antagonists were determined. All antagonists enhanced the stimulation-evoked overflow of tritium, i.e. the release of noradrenaline, indicating the existence of presynaptic alpha 2-autoreceptors. The EC30%, values of the antagonists (concentrations that increased the evoked overflow of tritium by 30%) were taken as a measure of affinity for the auto-receptors. The subclassification was based on a comparison of these affinity estimates with affinities for alpha 2A, alpha 2B, alpha 2C and alpha 2D radioligand binding sites and for previously classified alpha 2A- and alpha 2D-autoreceptors. The comparison indicates that the alpha 2-autoreceptors in the brain cortex of Cercopithecus aethiops belong to the genetic alpha 2A/D and not the alpha 2B or alpha 2C subtype. In their pharmacological properties, the autoreceptors are closest to the pharmacological alpha 2D subtype. The results support the notion that alpha 2-autoreceptors belong at least predominantly to the alpha 2A/D subtype of alpha 2-adrenoceptors.

In segments of human right atrial appendages preincubated with [3H]noradrenaline and superfused with physiological salt solution containing desipramine and corticosterone, we determined the effects of 5-hydroxytryptamine (5-HT) receptor agonists and antagonists on tritium overflow evoked by transmural electrical stimulation (2 Hz). Tritium overflow was inhibited by 5-HT, 5-carboxamidotryptamine (5-CT), 5-methoxytryptamine (5-MeOT), 5-methoxy-3(1,2,3,6-tetrahydro-4-pyridinyl)-1H indole succinate (RU 24969) and sumatriptan. Yohimbine and oxymetazoline (in the presence of idazoxan) also inhibited tritium overflow. The inhibitory potency of the drugs was significantly correlated with their affinity for 5-HTID receptors in human brain and for cloned human 5-HT1D alpha and 5-HT1D beta receptors, but not with their affinity for 5-HT1B, 5-HT1E, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3, 5-HT5A, 5-HT5B and 5-HT7 receptors. The potency order 5-CT > 5-HT > 5-MeOT is opposite to the order of affinities reported for 5-HT6 binding sites. The preferential 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetraline (up to 0.3 microM) and the selective 5-HT4 receptor agonist cisapride (up to 1 microM) failed to inhibit tritium overflow. L-694,247, a potent 5-HT1D beta receptor agonist, did not inhibit tritium overflow, but counteracted the inhibitory effect of 5-HT. Ketanserin at a concentration which should block 5-HT1D alpha but not 5-HT1D beta receptors and methiothepin at a concentration which may be assumed to block both 5-HT1D alpha and 5-HT1D beta receptors antagonized the inhibitory effect of 5-HT. Propranolol and ondansetron did not modify the 5-HT-induced inhibition of release. In conclusion, noradrenaline release in human right atrial appendages is inhibited via 5-HT receptors which are located on the noradrenergic axon terminals. These inhibitory presynaptic 5-HT receptors belong to the 5-HT1D subfamily. The ability of ketanserin to antagonize the inhibitory effect induced by activation of these receptors suggests that they can be subclassified as 5-HT1D alpha.

The excitatory junction current (EJC) evoked by electrical stimulation of postganglionic sympathetic nerves of rat tail artery with 100 pulses at 2 Hz, at 1.3 mmol/l external Ca2+, was used as a measure of the per pulse release of ATP. In controls the EJCs were initially facilitated, then gradually depressed during the stimulus train. The first EJC was slightly depressed by the alpha 2-adrenoceptor antagonist yohimbine, but starting from the 4th pulse the EJCs were enhanced. Yohimbine increased the early facilitation without markedly modifying the subsequent depression. The yohimbine-induced enhancement of EJCs caused by pulses 11-100 was, thus, constant. The noradrenaline reuptake blocker cocaine depressed the EJCs, abolished the early facilitation and slightly enhanced the depression. These effects of cocaine were reversed by further addition of yohimbine. The alpha 2-adrenoceptor agonist xylazine (1 and 10 mumol/l) dose dependently depressed the EJCs starting from the first pulse. The inhibitory effect of 1 mumol/l xylazine, but not that of 10 mumol/l xylazine, declined with train length. The inhibition of individual EJCs caused by activation of presynaptic alpha 2-adrenoceptors was used to monitor the concentration of released noradrenaline at these receptors. The ratio of individual EJCs in the presence and absence of yohimbine was assumed to reflect, pulse by pulse, the relative concentration of released noradrenaline at the presynaptic alpha 2-adrenoceptors, and hence termed [NA]alpha 2. For comparison, the concentration of endogenous noradrenaline was monitored electrochemically by differential pulse amperometry with a carbon fibre microelectrode; this signal is termed [NA]CF. [NA]alpha 2 and [NA]CF grew during the first 7-10 or 14-16 pulses, respectively, and then remained relatively constant throughout the stimulus train. Cocaine caused [NA]alpha 2 and [NA]CF to continue to grow during the first 35 and 50 pulses, and enhanced their peak levels by 180% and 320%, respectively. For comparison with the effects on the EJCs mediated via presynaptic alpha 2-adrenoceptors, those caused by varying external Ca2+ level were examined. At 0.65 mmol/l Ca2+ the amplitude of the first EJC was smaller than that at 1.3 mmol/l Ca2+, but the facilitation of later EJCs was enhanced and the subsequent depression reduced. An increase in external Ca2+ to 2.6 mmol/l had the opposite effects. All effects on EJCs caused by changes in external Ca2+ were maximal for the first EJC and then declined with the train length.(ABSTRACT TRUNCATED AT 400 WORDS)

Slices of the rabbit caudate nucleus, preincubated with [3H]dopamine and subjected to electrical field stimulation, were used (1) to investigate the involvement of G-proteins in the signal transduction of presynaptic D2 (auto)receptors and kappa-opioid receptors on dopaminergic axon terminals in this tissue and (2) to study a possible mutual interaction of these two presynaptic receptors. Pretreatment of the slices with either pertussis toxin (8 micrograms/ml; 18 h), or N-ethylmaleimide (30 microM, 30 min) significantly reduced the inhibitory effects of both the D2 agonist quinpirole and the kappa-opioid receptor agonist U-50488H on the [3H]overflow evoked by 36 pulses (2 ms, 24 mA, 0.3 Hz), suggesting the coupling of both receptors to G-proteins. Experiments designed to study possible interactions of these two presynaptic receptors were carried out under stimulation conditions (only 1 pulse), which strongly diminish interference of endogenous transmitters released in the tissue with modulatory effects of exogenous drugs. For instance, due to the presence of endogenous dopamine, quinpirole was much less potent during 36-pulse-than during 1-pulse field stimulation, whereas the D2 antagonist domperidone was almost without effect in the latter case. Using the 1-pulse stimulation paradigm, the concentration/response curve of quinpirole was unaffected in the presence of the half-maximal inhibitory concentration of U-50,488 H (0.1 microM). On the other hand, also quinpirole at its half-maximal inhibitory concentration (0.1 microM), hardly affected the concentration/response curve of U-50,488 H: only high concentrations of U-50,488 H (above 1 microM) seemed to be slightly less effective in the presence than in the absence of the D2 agonist.(ABSTRACT TRUNCATED AT 250 WORDS)

In superfused rat brain cortex slices and synaptosomes preincubated with [3H]noradrenaline the effect of agonists or antagonists at presynaptic H3 receptors on NMDA-evoked [3H]noradrenaline release was investigated. In experiments on slices, histamine and the preferential H3 receptor agonist R-(-)-alpha-methylhistamine inhibited NMDA-evoked tritium overflow (IC20 values 0.27 mumol/l or 0.032 mumol/l, respectively); S-(+)-alpha-methylhistamine (up to 10 mumol/l) as well as the selective H1 receptor agonist (2-(2-thiazolyl)ethylamine and the selective H2 receptor agonist dimaprit (each up to 10 mumol/l) were ineffective. The H3 receptor antagonist thioperamide abolished the inhibitory effect of histamine whereas the preferential H1 receptor antagonist dimetindene and the preferential H2 receptor antagonist ranitidine were ineffective. In experiments on synaptosomes, histamine and R-(-)-alpha-methylhistamine inhibited NMDA-evoked tritium overflow, whereas 2-(2-thiazolyl)ethylamine or dimaprit had no effect. The inhibitory effect of histamine was abolished by thioperamide. When tritium overflow was stimulated by NMDA in the presence of omega-conotoxin GVIA (which by itself decreased the response to NMDA by about 55%), R-(-)-alpha-methylhistamine did not inhibit NMDA-evoked overflow. It is concluded that NMDA-evoked noradrenaline release in the cerebral cortex can be modulated by inhibitory H3 receptors. NMDA receptors and H3 receptors are both located presynaptically and may interact at the same noradrenergic varicosity. An unimpaired function of the N-type voltage-sensitive calcium channel probably is a prerequisite for the inhibition of NMDA-evoked noradrenaline release by H3 receptor stimulation.

The effects of various opioid receptor agonists and antagonists were studied in rabbit caudate nucleus slices preincubated with either [3H]dopamine or [3H]choline, superfused with medium (containing in most experiments the D2 receptor antagonist domperidone) and subjected to electrical field stimulation. The stimulation-evoked [3H]overflow from slices prelabeled with [3H]dopamine (evoked [3H]dopamine release) was significantly reduced by preferential kappa-opioid receptor agonists, like U-50,488 H, but not by mu- or delta-opioid receptor selective drugs. Opioid receptor antagonists shifted the concentration/response curve of U-50,488 H to the right (apparent pA2-value of the kappa-selective antagonist nor-binaltorphimine: 10.1) and enhanced the evoked dopamine release in the presence of a mixture of peptidase inhibitors. On the other hand, the [3H]overflow from rabbit caudate nucleus slices prelabeled with [3H]choline (evoked acetylcholine release) remained almost unaffected by any opioid receptor agonist, as long as the presynaptic D2 heteroreceptor was blocked with domperidone: in the absence of domperidone, U-50,488 H exhibited facilitatory effects. For comparison, the effects of the preferential delta-opioid receptor agonist DPDPE was also studied in slices of the rat striatum, where it clearly inhibited the evoked acetylcholine release. From our data we conclude that in the rabbit caudate nucleus the evoked dopamine release is inhibited by both exogenous and endogenous opioids via presynaptic kappa-opioid receptors, whereas the evoked release of acetylcholine is not, or only indirectly (via released dopamine) affected by opioids.

The mechanism by which presynaptic muscarinic autoreceptors inhibit the release of acetylcholine (ACh) from cerebrocortical cholinergic fibres has not been clarified. To test the view that muscarinic autoreceptors act by decreasing Ca2+ influx, we performed experiments in which rat cerebrocortical prisms were preloaded with (14C)choline, washed, depolarized with 14-65 mM K+ in the absence of Ca2+ and then exposed (still under depolarization) to various concentrations of Ca2+ to evoke the release of (14C)ACh. The muscarinic agonist, oxotremorine, used at a 100 microM concentration, inhibited the release of (14C)ACh by 59-86% in experiments with 14 and 26.5 mM K+ but had no significant effect at 65.5 mM K+. No systematic changes in the inhibitory effects of oxotremorine could be found at any of the K+ concentrations used when the concentration of Ca2+ was varied in the range of 0.25-4.0 mM. At 2 mM Ca2+ and K+ concentrations above 14 mM, the inhibitory effect of oxotremorine was inversely related to the concentration of K+. The inhibitory effect of oxotremorine on (14C)ACh release was not blocked by 100 microM 4-amino-pyridine. The fact that the inhibitory effect of oxotremorine could not be overcome by an increase in the concentration of Ca2+ suggests that, under the conditions used, a restriction of the influx of Ca2+ did not play a major role in the muscarinic inhibition of ACh release; rather, oxotremorine appeared to act by decreasing membrane depolarization.2+ of the Ca(2+)-voltage hypothesis of neurotransmitter release, supposing