Effects of Cyclic AMP Analogues and Phosphodiesterase Inhibitors on K+-Induced [3H]Noradrenaline Release from Rat Brain Slices and on Its Presynaptic α-Adrenergic Modulation

The possible role of cyclic AMP in the presynaptic alpha-adrenoceptor-mediated modulation of [3H]noradrenaline (NA) release induced by 13 mM K+ from superfused rat cerebral cortex slices was investigated. Both dibutyryl-cyclic AMP (db-cAMP) and 8-bromo-cyclic AMP (8-Br-cAMP) dose-dependently (10(-4) - 10(-2) M) enhanced K+-induced (3H]NA release, maximally to about 160% of control. In contrast, db-cAMP had no effect on calcium-induced [3H]NA release in the presence of the calcium ionophore A 23187. Surprisingly, the phosphodiesterase (PDE) inhibitors 3-isobutyl-1-methylxanthine (IBMX). 7-benzyl-IBMX, 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (ZK 62771), and 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724) appeared to inhibit K+-induced [3H]NA release in a dose-dependent (10(-5) - 10(-3) M) manner. At a concentration of 10(-4) M, AK 62771 caused an inhibition of [3H]NA release by 30%, and this inhibitory effect was not affected by 10(-6) M phentolamine nor by 10(-3) M db-cAMP or 10(-4) M theophylline. Theophylline by itself enhanced [3H]NA release to about 135% of control. The inhibitor effect of the alpha-adrenoceptor agonist oxymetazoline (1 micro M) and the enhancing effect of the antagonist phentolamine (1 micro M) on [3H]NA release were significantly decreased in the presence of 10(-3) M db-cAMP or 8-Br-cAMP, whereas 10(-4) M ZK 62771 had no effect. In the presence of 10(-2) M NaF, a potent activator of adenylate cyclase, the inhibitory effect of oxymetazoline (1 micro M) on [3H]NA release was significantly decreased. The data obtained with the cyclic AMP analogues support the hypothesis that activation of presynaptic alpha-receptors modulating NA release results in an inhibition of a presynaptic adenylate cyclase. Possible causes for the anomalous effects of th PDE inhibitors are discussed.     

Role of Adenylate Cyclase in Presynaptic α2-Adrenoceptor- and μ-Opioid Receptor-Mediated Inhibition of [3H]Noradrenaline Release from Rat Brain Cortex Slices

Rat brain cortex slices, prelabelled with [3H]noradrenaline, were superfused and exposed to electrical biphasic block pulses (1 Hz; 12 mA, 4 ms) or to the Ca2+ ionophore A 23187 (10 microM) in the presence of 1.2 mM Ca2+. Forskolin (10 microM), 8-bromo-cyclic AMP (300 microM), and dibutyryl-cyclic AMP (300 microM) facilitated both the electrically evoked and A 23187-induced [3H]noradrenaline release, whereas the phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine (IBMX, 300 microM) and 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (ZK 62771, 30 microM) enhanced the electrically evoked release only. The inhibitory effects of clonidine (1 nM-1 microM) and the facilitatory effect of phentolamine (0.01-10 microM) on the electrically evoked [3H]noradrenaline release were strongly reduced in the presence of 8-bromo-cyclic AMP. Clonidine (1 microM) reduced and phentolamine (3 microM) enhanced A 23187-induced [3H]noradrenaline release, provided that the slices were simultaneously exposed to forskolin. The inhibitory effects of morphine (1 microM) and [D-Ala2-D-Leu5]enkephalin (DADLE, 0.3 microM), like that of the Ca2+ antagonist Cd2+ (15 microM), on the electrically evoked release of [3H]noradrenaline were not affected by 8-bromo-cyclic AMP. Moreover, morphine and DADLE did not inhibit A 23187-induced release in the absence or presence of forskolin. These data strongly suggest that in contrast to presynaptic mu-opioid receptors, alpha 2-adrenoceptors on noradrenergic nerve terminals are negatively coupled to adenylate cyclase and may thus reduce neurotransmitter release by inhibiting the feed-forward action of cyclic AMP on the secretion process.     

Lack of Selectivity Between the Uptake of [3H] Adrenaline and [3H]Noradrenaline into Rat Hypothalamic Slices

The uptake of [3H]adrenaline and [3H]noradrenaline into rat hypothalamic slices was compared for determination of whether adrenaline uptake was independent of uptake into noradrenergic neurones. Kinetic analysis revealed a similar high-affinity uptake process for both adrenaline and noradrenaline, with Km and Vmax values within similar ranges. These uptakes were inhibited by desipramine and maprotiline in a dose-dependent manner, but the selective dopamine and 5-hydroxytryptamine uptake inhibitors benztropine and fluoxetine, respectively, were without effect. Competition for uptake sites by unlabelled adrenaline with [3H]adrenaline and [3H]-noradrenaline and by unlabelled noradrenaline with [3H]-adrenaline and [3H]noradrenaline was very similar. Lesioning of the major adrenaline-containing cell group (C1 cell group) decreased the hypothalamic adrenaline concentration but had no effect on hypothalamic [3H]adrenaline or [3H]noradrenaline uptake. The results suggest that exogenous adrenaline is largely taken up by high-affinity sites on noradrenergic nerve terminals.     

4-Aminopyridine Stimulates B-50 (GAP43) Phosphorylation and [3H]Noradrenaline Release in Rat Hippocampal Slices

In situ phosphorylation of the presynaptic protein kinase C substrate B-50 was investigated in rat hippocampal slices incubated with the convulsant drug 4-aminopyridine (4-AP). Phosphorylation of B-50 was significantly enhanced 1 min after the addition of 4-AP (100 microM). This increase by 4-AP was concentration dependent (estimated EC50 30-50 microM). Concomitant with the changes in B-50 phosphorylation, 4-AP also dose-dependently stimulated [3H]noradrenaline [( 3H]NA) release from the slices. 4-AP stimulated [3H]NA release within 5 min to seven times the control level. The B-50 phosphorylation induced by 4-AP remained elevated after removal of the convulsant, this is contrast to B-50 phosphorylation induced by depolarization with K+. A similar persistent increase was observed for [3H]NA release after a 5-min incubation period with 4-AP. These results give more insight into the molecular mechanisms underlying 4-AP-induced epileptogenesis and provide further evidence for the correlation between B-50 phosphorylation and neurotransmitter release in the hippocampal slice.     

Effect of Chronic Lithium Treatment on 5-Hydroxytryptamine Autoreceptors and Release of 5-[3H]Hydroxytryptamine from Rat Brain Cortical, Hippocampal, and Hypothalamic Slices

The effect of acute and chronic lithium treatments on 5-hydroxytryptamine (5-HT, serotonin) release and on its regulation by presynaptic 5-HT autoreceptors was studied in [3H]5-HT preloaded superfused rat brain slices. The [3H]5-HT overflow evoked by a 30-s exposure to 65 mM K+ was increased after 3 weeks of ingestion of lithium-containing diet in the three brain areas examined. Acute injection of 4 mEq/kg lithium chloride did not affect 5-HT release. The K+-induced release observed in both control and chronically lithium-treated animals was Ca2+-dependent. Chronic lithium treatment was also found to be associated with a decrease in basal [3H]5-HT overflow in the cortex and hypothalamus but not in hippocampus [corrected]. The Ca2+-independent overflow induced by fenfluramine was also decreased in cortical slices from lithium-treated animals. The sensitivity of the inhibitory 5-HT autoreceptors was assessed by the response to the 5-HT agonist 5-methoxytryptamine. The results indicate a marked reduction in the maximal inhibition of [3H]5-HT release induced by 5-methoxytryptamine in slices obtained from animals which have been treated with lithium for 3 weeks. These data suggest that the functional down regulation of the prejunctional 5-HT sites may be responsible for the increase in K+-stimulated 5-HT overflow in brain slices of animals treated chronically with lithium.     

Effect of Potassium on the Release of [3H]Noradrenaline from Rabbit and Human Pulmonary Artery

The release of [3H]noradrenaline ( [3H]NA) from rabbit and human isolated pulmonary artery has been measured. Removal of external potassium ions enhanced both the resting and stimulated release of [3H]NA from the strips. On adding K+ to tissues which had been suspended in K+-free Krebs solution, the release of [3H]NA was reduced in both stimulated and unstimulated tissues. Selective inhibition of presynaptic alpha 2-adrenoceptors by yohimbine significantly potentiated the release of [3H]NA evoked by stimulation in K+-free solution. The presynaptic inhibitory effect of NA was much less pronounced when the release was enhanced by the removal of external K+. Since the activity of NA, K-ATPase may be affected by removing K+ or by adding it to tissue previously kept in K+-free solution, the results may indicate involvement of the sodium pump in NA release.     

Hypoxia Enhances [3H]Noradrenaline Release Evoked by Nicotinic Receptor Activation from the Human Neuroblastoma SH-SY5Y

Abstract: We have used the human sympathetic neuronal line SH-SY5Y to investigate the effects of hypoxia on noradrenaline (NA) release evoked by either raised [K⁺]_o (100 m M ) or the nicotinic acetylcholine receptor (nAChR) agonist dimethylphenylpiperazinium iodide (DMPP). NA release was monitored by loading cells with [³H]NA and collecting effluent fractions from perfused cells kept in a sealed perifusion chamber. Cells were challenged twice with either stimulus and release was expressed as that evoked by the second challenge as a fraction of that evoked by the first. K⁺-evoked release was unaffected by hypoxia (P o ₂≅ 30–38 mm Hg), but release evoked by DMPP was significantly increased. For both stimuli, replacement of Ca²⁺_o with 1 m M EGTA abolished NA release. K⁺-evoked release was also dramatically reduced in the presence of 200 µ M Cd²⁺ to block voltage-gated Ca²⁺ channels, but DMPP-evoked release was less affected. In hypoxia, DMPP-evoked Cd²⁺-resistant NA release was dramatically increased. Our findings indicate that hypoxia increases NA release evoked from SH-SY5Y cells in response to nAChR activation by increasing Ca²⁺ influx through the nAChR pore, or by activating an unidentified Cd²⁺-resistant Ca²⁺-influx pathway. As acetylcholine is the endogenous transmitter at sympathetic ganglia, these findings may have important implications for sympathetic activity under hypoxic conditions.     

α2-Adrenoceptor-Mediated Inhibition of Electrically Evoked [3H]Noradrenaline Release from Chick Sympathetic Neurons: Role of Cyclic AMP

Abstract: This study explores the role of cyclic AMP in electrically evoked [³H]noradrenaline release and in the α₂-adrenergic modulation of this release in chick sympathetic neurons. Along with an increase in stimulation-evoked tritium overflow, applications of forskolin enhanced the formation of intracellular cyclic AMP. Both effects of forskolin were potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. The forskolin-induced increase in overflow was abolished by the Rp-diastereomer of cyclic AMP-thioate, an antagonist at cyclic AMP-dependent protein kinases, and 1,9-dideoxy-forskolin, an inactive analogue at adenylyl cyclase, had no effect on the evoked overflow. A 24-h pretreatment with either cholera toxin or forskolin reduced the subsequent forskolin-induced accumulation of cyclic AMP and inhibited the stimulation-evoked release. Basal cyclic AMP production, however, remained unaltered after forskolin treatment and was enhanced after 24 h of cholera toxin exposure. The α₂-adrenergic agonist bromoxidine did not affect the formation of cyclic AMP stimulated by forskolin but reduced electrically evoked release. However, effects of bromoxidine on ³H overflow were attenuated by forskolin as well as by 8-bromo-cyclic AMP. Effects of bromoxidine on [³H]noradrenaline release were paralleled by an inhibition of voltage-activated Ca²⁺ currents, primarily through a delayed time course of current activation. This effect was abolished when either forskolin or 8-bromo-cyclic AMP was included in the pipette solution. Both substances, however, failed to affect Ca²⁺ currents in the absence of bromoxidine. These results suggest that the signaling cascade of the α₂-adrenergic inhibition of noradrenaline release involves voltage-activated Ca²⁺ channels but not cyclic AMP. Elevated levels of cyclic AMP, however, antagonize this α₂-adrenergic reduction, apparently through a disinhibition of Ca²⁺ channels.     

Effect of Inhibitors of Eicosanoid Metabolism on Release of [3H]Noradrenaline from the Human Neuroblastoma, SH-SY5Y

Abstract: Nordihydroguaiaretic acid (NDGA; a lipoxygenase inhibitor), LY-270766 (an inhibitor of 5-lipoxygenase), and the diacylglycerol lipase inhibitor RG 80267 completely eliminated potassium-evoked release of [³H]noradrenaline ([³H]NA) from the human neuroblastoma clone SH-SY5Y with IC₅₀ values of 10, 15, and 30 μ M , respectively. In contrast, these inhibitors only partially inhibited carbachol-evoked release and had little effect on the calcium ionophore A23187-evoked release of NA in this cell line. Arachidonic acid partially inhibited potassium- and A23187-evoked release but did not reverse the inhibition of potassium-evoked release observed in the presence of RG 80267. These studies suggest that arachidonic acid (or its lipoxygenase products) are not important intermediates in the regulation of exocytosis in SH-SY5Y. This conclusion is strengthened by our studies in which SH-SY5Y cells were grown in medium supplemented with bovine serum albumin-linoleic acid (50 μ M ). Under these conditions there was a selective increase in content of membrane polyunsaturated fatty acids of the ω6 series, including arachidonic acid; however, these changes did not effect potassium-, veratridine-, carbachol-, or calcium ionophoreevoked release of [³H]NA.     

Mobilization of Inositol 1,4,5-Trisphosphate-Sensitive Ca2+ Stores Supports Bradykinin- and Muscarinic-Evoked Release of [3H]Noradrenaline from SH-SY5Y Cells

Abstract: The human neuroblastoma cell line SH-SY5Y, maintained at confluence for 14 days, released [³H]-noradrenaline ([³H]NA) when stimulated with either the muscarinic receptor agonist methacholine or bradykinin. The major fraction of release was rapid, occurring in <10 s, whereas nicotine-evoked release was slower. When the extracellular [Ca²⁺] ([Ca²⁺]_e) was buffered to ～50–100 nM, release evoked by nicotine was abolished, whereas that in response to methacholine or bradykinin was reduced by ～50% with EC₅₀ values of ?5.46 ± 0.05 M and ?7.46 ± 0.06 M (log₁₀), respectively. Methacholine and bradykinin also produced rapid elevations of both inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] and intracellular free [Ca²⁺] ([Ca²⁺]_i). These elevations were reduced at low [Ca²⁺]_e and under these conditions the EC₅₀ values for peak elevation of [Ca²⁺]_i were ?6.00 ± 0.14 M for methacholine and ?7.95 ± 0.34 M for bradykinin (n = 3 for all EC₅₀ determinations). At low [Ca²⁺]_e, depletion of nonmitochondrial intracellular Ca²⁺ stores with the Ca²⁺-ATPase inhibitor thapsigargin produced a transient small elevation of [Ca²⁺]_i and a minor release of [³H]NA. At low [Ca²⁺]_e, thapsigargin abolished elevation of [Ca²⁺]_i in response to methacholine and bradykinin and completely inhibited their stimulation of [³H]NA release. It is proposed, therefore, that Ca²⁺ release from Ins(1,4,5)P₃-sensitive stores is a major trigger of methacholine- and bradykinin-evoked [³H]NA release in SH-SY5Y cells.     

Roles for Nitric Oxide as an Intra- and Interneuronal Messenger at NMDA Release-Regulating Receptors: Evidence from Studies of the NMDA-Evoked Release of [3H]Noradrenaline and d-[3H]Aspartate from Rat Hippocampal Slices

Abstract: N-Methyl-d -aspartate (NMDA) receptors regulating the release of [³H]noradrenaline ([³H]NA) and d -[³H]aspartate (d -[³H]Asp) were investigated in superfused slices of rat hippocampus in the presence and absence of nitrergic drugs to examine a possible role for nitric oxide (NO) in the release process. In Mg²⁺-free Krebs-Henseleit buffer, the NMDA-evoked release of [³H]NA and d -[³H]Asp was Ca²⁺ dependent and inhibited by the NMDA antagonist (±)-3-(2-carboxypiperazin-4-yl)propenyl-1-phosphonic acid. NMDA-stimulated release of [³H]NA was tetrodotoxin (TTX; 0.1–2 µM) sensitive, whereas that for d -[³H]Asp was TTX insensitive, indicating that the NMDA receptors involved are differentially localized; those for d -[³H]Asp appear to be presynaptic, whereas those for [³H]NA are extrasynaptic in location. l -Arginine (100 µM), the natural precursor of NO synthesis, enhanced NMDA-evoked release of [³H]NA (100%) and d -[³H]Asp (700%). Exogenous NO donors—sodium nitroprusside, 3-morpholinosyndnomine, and S-nitroso-N-acetylpenicillamine (all 100 µM)—stimulated the NMDA-evoked release. An exception was the inhibition by nitroprusside of NMDA-evoked release of [³H]NA, where the presence of antioxidants may influence channel activity. Inhibitors of NO synthase (N^G-nitro-, N^G-methyl-, and N^G-amino-l -arginine, all 100 µM) attenuated (50–80%) the NMDA-stimulated release of [³H]NA and d -[³H]Asp, as did KN-62 (10 µM), a specific inhibitor of calmodulin kinase II. Our data support roles for the NO transducing system subsequent to the activation of NMDA release-regulating receptors as both an intraneuronal (presynaptically) and an extraneuronal messenger.     

Effects of Antidepressant Drug Treatments on α2-Adrenoceptor Control of [3H]Noradrenaline Release from Hypothalamic Synaptosomes

KCl (16 mM) stimulated the release of [3H]noradrenaline ([3H]NA) from rat hypothalamic synaptosomes in a Ca2+-dependent manner; this release was attenuated by clonidine (0.01-100 microM). Changes in the release of [3H]NA and the functional status of alpha 2-adrenoceptors in the medial hypothalamus of rats treated acutely and chronically with clorgyline (1 mg/kg/day) or desipramine (DMI, 10 mg/kg/day) were assessed using superfused synaptosomes in which the attenuating effects of clonidine (1 microM) or the potentiating effects of yohimbine (1 microM) on K+-evoked release of [3H]NA were measured. After acute administration of DMI, significantly less [3H]NA was accumulated into synaptosomes. Although total (spontaneous + K+-evoked) [3H]NA release from these synaptosomes was unchanged, a significant reduction was apparent in the K+-evoked release from the DMI-treated tissue. Attenuation of K+-evoked release by clonidine was abolished in both these acute treatment groups. Following the chronic antidepressant drug regimens, [3H]NA uptake into DMI-treated tissue remained significantly reduced although total percent and K+-evoked [3H]NA release were unchanged. The K+-evoked release of [3H]NA in S1 was significantly enhanced (by 22%) in the clorgyline treatment group. Attenuation of K+-evoked [3H]NA release by clonidine in both chronic antidepressant-treated tissues was not significantly changed. It is concluded that the functional sensitivity of alpha 2-adrenoceptors on nerve endings in the medial hypothalamus is unchanged by these chronic antidepressant drug regimens. In synaptosomes from untreated tissue, yohimbine significantly potentiated K+-evoked release of [3H]NA; this effect was unchanged after acute regimens and reduced after chronic administration of both the antidepressants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potassium- and Carbachol-Evoked Release of [3H]Noradrenaline from Human Neuroblastoma Cells, SH-SY5Y

The human neuroblastoma clone SH-SY5Y expresses potassium-, carbachol-, and calcium ionophore A23187-evoked, calcium-dependent release of [3H]noradrenaline. Release in response to carbachol and potassium was greater than additive. Atropine (Ki = 0.33 nM), hexahydrosiladifenidol (Ki = 18 nM), and pirenzepine (Ki = 1,183 nM) completely inhibited the carbachol-evoked noradrenaline release, an order of potency suggesting that an M3 receptor was linked to release. In contrast, noradrenaline release was only partially inhibited by the M2-selective antagonists methoctramine (10(-4) M) and AFDX-116 (10(-4) M), by approximately 14 and 46%, respectively. The nicotinic antagonist d-tubocurarine (10(-4) M) resulted in a partial inhibition of release, a finding suggesting that a nicotinic receptor may also be involved. SH-SY5Y provides a suitable cell line in which to study the biochemical mechanisms underlying the cholinergic receptor regulation of noradrenaline release.     

Stimulation of [3H]GABA and β-[3H]Alanine Release from Rat Brain Slices by cis-4-Aminocrotonic Acid

Abstract: cis -4-Aminocrotonic acid (CACA; 100 µ M ), an analogue of GABA in a folded conformation, stimulated the passive release of [³H]GABA from slices of rat cerebellum, cerebral cortex, retina, and spinal cord and of β-[³H]alanine from slices of cerebellum and spinal cord without influencing potassium-evoked release. In contrast, CACA (100 µ M ) did not stimulate the passive release of [³H]taurine from slices of cerebellum and spinal cord or of d -[³H]aspartate from slices of cerebellum and did not influence potassium-evoked release of [³H]taurine from the cerebellum and spinal cord and d -[³H]aspartate from the cerebellum. These results suggest that the effects of CACA on GABA and β-alanine release are due to CACA acting as a substrate for a β-alanine-sensitive GABA transport system, consistent with CACA inhibiting the uptake of β-[³H]alanine into slices of rat cerebellum and cerebral cortex. The observed K _i for CACA against β-[³H]alanine uptake in the cerebellum was 750 ± 60 µ M . CACA appears to be 10-fold weaker as a substrate for the transporter system than as an agonist for the GABA_c receptor. The effects of CACA on GABA and β-alanine release provide indirect evidence for a GABA transporter in cerebellum, cerebral cortex, retina, and spinal cord that transports GABA, β-alanine, CACA, and nipecotic acid that has a similar pharmacological profile to that of the GABA transporter, GAT-3, cloned from rat CNS. The structural similarities of GABA, β-alanine, CACA, and nipecotic acid are demonstrated by computer-aided molecular modeling, providing information on the possible conformations of these substances being transported by a common carrier protein.     

Stress-Induced Enhancement of Suppression of [3H]GABA Release from Striatal Slices by Presynaptic Autoreceptor

The effect of cold and immobilization stress on presynaptic GABAergic autoreceptors was examined using the release of [3H]GABA (gamma-aminobutyric acid) from slices of rat striatum. It was found that in vitro addition of delta-aminolevulinic acid, as well as GABA agonists such as muscimol and imidazoleacetic acid, exhibited a significant suppression of the striatal release of [3H]GABA evoked by the addition of high potassium, whereas delta-aminovaleric acid had no significant effects on the evoked release. These suppressive actions were antagonized invariably by the GABA antagonists, bicuculline and picrotoxin, but not by the glycine antagonist, strychnine. Cholinergic agonists, such as pilocarpine and tetramethylammonium, also attenuated significantly the evoked release of [3H]GABA from striatal slices, while none of its antagonists, including atropine, hexamethonium and d-tubocurarine, affected the release. On the other hand, in vitro addition of dopamine receptor agents such as dopamine, apomorphine, and haloperidol, or the inhibitory amino acids, glycine, beta-alanine, and taurine failed to influence the evoked release of [3H]GABA from striatal slices. Application of a cold and immobilization stress for 3 h was found to induce a significant enhancement of the suppressive effects by muscimol and delta-aminolevulinic acid on the evoked release of [3H]GABA, without affecting that by pilocarpine and tetramethylammonium. These results suggest that the release of GABA from striatal GABA neurons may be regulated by presynaptic autoreceptors for this neuroactive amino acid, and may play a significant functional role in the exhibition of various symptoms induced by stress.     

Cyclothiazide Selectively Potentiates AMPA- and Kainate-Induced [3H]Norepinephrine Release from Rat Hippocampal Slices

Abstract: Activation of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) subtype of ionotropic glutamate receptors has been shown to result in a rapid desensitization of the receptor in the presence of certain agonists. One effect of AMPA receptor desensitization in the hippocampus may be to decrease the efficacy of AMPA receptor agonists at stimulating the release of norepinephrine from noradrenergic terminals. Recently, cyclothiazide was reported to inhibit AMPA receptor desensitization by acting at a distinct site on AMPA receptors. We have examined the effect of cyclothiazide on AMPA- and kainate (KA)-induced norepinephrine release from rat hippocampal slices to determine whether cyclothiazide would increase the efficacy of AMPA-induced [³H]norepinephrine release by inhibiting AMPA receptor desensitization. Cyclothiazide was observed to potentiate markedly both AMPA- and KA-induced [³H]norepinephrine release. This potentiation is selective for AMPA/KA receptors as cyclothiazide did not potentiate N -methyl- d -aspartate-induced [³H]norepinephrine release or release induced by the nonspecific depolarizing agents veratridine and 4-aminopyridine. These results demonstrate that AMPA receptor-mediated modulation of [³H]norepinephrine release from rat brain slices is a useful approach to studying the cyclothiazide modulatory site on the AMPA receptor complex.     

The Synthesis and Release of [3H-Tyrosine1]Methionine5-Enkephalin from Guinea Pig Brain Slices

Abstract: Stores of methionine-enkephalin were labelled on the N -terminal by incubation of whole brain slices with [³H]tyrosine (10 °Ci/ml). The ³H radioactivity corresponding to the position of authentic Met-enkephalin after extraction on Amberlite XAD₂ and separation by thin-layer chromatography was taken as an index of synthesis. Maximal incorporation of the labelled tyrosine into Met-enkephalin was attained after 4 h of incubation at 37°C and was inhibited in the presence of 10 μ M cycloheximide. Isolated nerve terminals failed to incorporate any [³H]tyrosine. The labelled compound had opiatelike activity and consisted of the same five amino acids as an authentic standard. Incubations with leucine aminopeptidase indicated that the labelled tyrosine was on the N -terminus and removal of this tyrosine resulted in loss of opiate-like activity. The incorporation of [¹⁴C]glycine, selected as an alternative precursor, was consistent with de novo synthesis and not N -terminal exchange. A radioimmunoassay was also used to quantify the amount of labelled Met-enkephalin. KCl (50 m M ) elicited a Ca²⁺-dependent release of the synthesised [³H]Met-enkephalin from whole brain slices and also from isolated nerve terminals. The release of Met-enkephalin radioimmunoactivity paralleled that of [³H]met-enkephalin. Preliminary investigations have suggested that carbamyl choline inhibited this release and its effect was partially reversed by atropine.     

On the Recovery of [3H]Noradrenaline from Different Metabolic Compartments of Rat Brain with Respect to the Role of Catechol-O-Methyltransferase

Abstract: Rats were treated with reserpine, desmethylimipramine, or carrier, either alone or in combination with tropolone. Either 10 min (t₁) or 1 h (t₂) after intraventricular injection of [³H]noradrenaline, they were decapitated. The total ³H activity and the recovery of [³H]noradrenaline were determined in tissue extracts from various brain regions. Maximum total ³H activity was measured at t₁ in all tropolone-treated rats; the mean sum of these results served as an estimate of the initial tissue concentration of [³H]noradrenaline. At t₁, 40–50% of the sum of [³H]noradrenaline and its metabolites was recovered unchanged in normal rats; reserpine and DMI reduced the recovery to 18–27%. In all groups, the decline of [³H]noradrenaline was retarded after t₁. Inhibition of catechol-O-methyltransferase by tropolone caused consistently elevated [³H]noradrenaline levels, but did not affect the metabolic rate after t₁ when compared with similarly pretreated, but tropolone-free rats. Thus, if catechol-O-methyltransferase was inhibited during the injection of [³H]noradrenaline, a higher percentage of the amine had been taken up into spaces with a slow noradrenaline turnover. The maximum increase was seen when the neuronal uptake, was inhibited by desmethylimipramine. This supported the hypothesis that an additional extraneuronal space exists, in addition to the known intraneuronal and extraneuronal compartments, which has a slow noradrenaline turnover. The tropolone effect on the noradrenaline recovery possibly shows that there might be a saturable “methylating system,” similar to that described for the periphery, in which catechol-O-methyltransferase is linked to the extraneuronal uptake₂. By affecting the access of noradrenaline to non-neuronal cells it might influence the rate of noradrenaline elimination from the intercellular space.     

Morphine and Enkephalins Potently Inhibit [3H]Noradrenaline Release from Rat Brain Cortex Synaptosomes: Further Evidence for a Presynaptic Localization of μ-Opioid Receptors

Synaptosomes prepared from rat cerebral cortex and labeled with [3H]noradrenaline (NA) were superfused with calcium-free Krebs-Ringer-bicarbonate medium and exposed to 10 mM K+ plus 0.1 mM Ca2+ so that [3H]NA release was induced. 6,7-Dihydroxy-N,N-dimethyl-2-aminotetralin (TL-99) strongly inhibited synaptosomal K+-induced [3H]NA release (EC50 = 5-10 nM) by activating alpha 2-adrenoceptors. Release was also inhibited (maximally by 40-50%) by morphine (EC50 = 5-10 nM), [Leu5]enkephalin (EC50 = approximately 300 nM), [D-Ala2,D-Leu5]enkephalin (DADLE), and Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol (DAGO) (EC50 values = approximately 30 nM). In contrast to the mu-selective opioid receptor agonists morphine and DAGO, the highly delta-selective agonist [D-Pen2,D-Pen5]enkephalin (1 microM) did not affect [3H]-NA release. Furthermore, the inhibitory effect of DADLE, an agonist with affinity for both delta- and mu-opioid receptors, was antagonized by low concentrations of naloxone. The findings strongly support the view that, like alpha 2-adrenoceptors, mu-opioid receptors mediating inhibition of NA release in the rat cerebral cortex are localized on noradrenergic nerve terminals.