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.     

μ-Opioid Receptors Mediate the Inhibitory Effect of Opioids on Dopamine-Sensitive Adenylate Cyclase in Primary Cultures of Rat Neostriatal Neurons

The receptors mediating the inhibition of D1 dopamine receptor-stimulated adenylate cyclase by opioids were examined in primary cultures of rat neostriatal neurons. Adenylate cyclase activity was dose-dependently increased by the selective D1 dopamine receptor agonist SKF 38393 (EC50 = 0.05 microM). This stimulation was fully antagonized by the selective D1 dopamine receptor antagonist SCH 23390 (1 microM). SKF 38393 (1 microM)-stimulated adenylate cyclase activity was strongly reduced (by almost 60%) by the highly selective mu-agonist [D-Ala2, MePhe4, Gly-ol5]-enkephalin (DAGO; EC50 = 0.006 microM) and high concentrations of the selective delta-agonist [D-Ser2(O-tert-butyl), Leu5]-enkephalyl-Thr6 (DSTBU-LET; EC50 = 0.13 microM) but not by the selective delta-agonist [D-penicillamine2, D-penicillamine5]enkephalin (DPDPE). D1 dopamine receptor-stimulated adenylate cyclase activity was also slightly reduced (by approximately 20%) by high concentrations of the kappa-agonist U50,488 (EC50 = 0.63 microM). The inhibitory effects of submaximally effective concentrations of DAGO, DSTBULET, and U50,488 were equally well antagonized by the mu-opioid receptor-selective antagonist naloxone (EC50 of approximately 0.1 microM). Neither the irreversible delta-ligand fentanyl isothiocyanate (1 microM) nor the reversible delta-antagonist ICI 174864 (1 microM) reversed the inhibitory effects of DSTBULET. The inhibitory effects of DAGO and U50,488 were equally well reversed by high concentrations (greater than 0.1 microM) of the kappa-opioid receptor-selective antagonist norbinaltorphimine. The effect of DAGO (1 microM) was already detectable after 1 day in culture, whereas DPDPE (1 microM) had no effect even after 28 days in culture. These data indicate that an homogeneous population of mu-opioid receptors coupled as inhibitors to D1 dopamine receptor-stimulated adenylate cyclase is expressed in rat neostriatal neurons in primary culture.     

Depolarization-induced release of [3H]histamine by high potassium concentrations,electrical stimulation and veratrine from rat brain slices after incubation with the radiolabelled amine

Brain slices obtained from neocortex, hypothalamus or hippocampus were incubated with [³H]histamine and subsequently superfused and exposed to different depolarizing stimuli, viz. high K⁺-concentrations, electrical field stimulation and veratrine. K⁺-induced release of tritium was completely calcium-dependent and its magnitude depended on the K⁺-concentration, with maximal release being reached at 56 mM K⁺. Electrically-evoked release of tritium increased with increasing frequencies and reached its maximum at about 20 Hz. The electrically-evoked release appeared to be totally calcium-dependent and it was strongly inhibited by tetrodotoxin. Veratrine (5–100 μM) also induced a release of tritium; maximal release was obtained at 100 μM veratrine. Veratrine-induced release was partially calcium-dependent and was strongly reduced by tetrodotoxin.Taken together the data indicate that the depolarization-induced release of tritium from brain slices pre-labelled with [³H]histamine, represents [³H]histamine release from neurons and not from either mast cells or glial cells. It remains to be established whether these neurons are specifically histaminergic.     

Differential effects of chronic agonist administration on mu-opioid receptor- and muscarinic receptor-regulated adenylate cyclase in rat striatal neurons.

In cultured rat striatal neurons exposed to 10 microM morphine or oxotremorine for 24 hours, we observed an increased (about 30%) dopamine D1 receptor-stimulated cyclic AMP production, whereas no desensitization of mu-opioid receptor or muscarinic cholinergic receptor was found. However, whereas upregulation of dopamine D1 receptor-stimulated adenylate cyclase activity upon 7 days morphine exposure was at least as pronounced as observed after 24 hours of exposure to the opioid, this adaptive phenomenon was virtually absent following one week of oxotremorine treatment. This reduced adenylate cyclase sensitization upon 7 days oxotremorine exposure appeared to coincide with desensitization of muscarinic cholinergic receptors. A possible role of the resistance of mu receptors to desensitization and the (resulting) upregulation of the neuronal adenylate cyclase system upon chronic receptor activation in the development of opiate tolerance and dependence is suggested.     

Effects of amphetamine on dopamine release in the rat nucleus accumbens shell region depend on cannabinoid CB1 receptor activation

The psychostimulant drug amphetamine is often prescribed to treat Attention-Deficit/Hyperactivity Disorder. The behavioral effects of the psychostimulant drug amphetamine depend on its ability to increase monoamine neurotransmission in brain regions such as the nucleus accumbens (NAC) and medial prefrontal cortex (mPFC). Recent behavioral data suggest that the endocannabinoid system also plays a role in this respect. Here we investigated the role of cannabinoid CB1 receptor activity in amphetamine-induced monoamine release in the NAC and/or mPFC of rats using in vivo microdialysis. Results show that systemic administration of a low, clinically relevant dose of amphetamine (0.5mg/kg) robustly increased dopamine and norepinephrine release (to ～175-350% of baseline values) in the NAC shell and core subregions as well as the ventral and dorsal parts of the mPFC, while moderately enhancing extracellular serotonin levels (to ～135% of baseline value) in the NAC core only. Although systemic administration of the CB1 receptor antagonist SR141716A (0-3mg/kg) alone did not affect monoamine release, it dose-dependently abolished amphetamine-induced dopamine release specifically in the NAC shell. SR141716A did not affect amphetamine-induced norepinephrine or serotonin release in any of the brain regions investigated. Thus, the effects of acute CB1 receptor blockade on amphetamine-induced monoamine transmission were restricted to dopamine, and more specifically to mesolimbic dopamine projections into the NAC shell. This brain region- and monoamine-selective role of CB1 receptors is suggested to subserve the behavioral effects of amphetamine.     

The Cell Wall of Fusarium oxysporum

Sugar analysis of isolated cell walls from three formae speciales of Fusarium oxysporum showed that they contained not only glucose and (N-acetyl)-glucosamine, but also mannose, galactose, and uronic acids, presumably originating from cell wall glycoproteins. Cell wall glycoproteins accounted for 50–60% of the total mass of the wall. X-ray diffraction studies showed the presence of α-1,3-glucan in the alkali-soluble cell wall fraction and of β-1,3-glucan and chitin in the alkali-insoluble fraction. Electron microscopy and lectin binding studies indicated that glycoproteins form an external layer covering an inner layer composed of chitin and glucan.     

Intermittent administration of morphine alters protein expression in rat nucleus accumbens

Repeated exposure to drugs of abuse causes time-dependent neuroadaptive changes in the mesocorticolimbic system of the brain that are considered to underlie the expression of major behavioral characteristics of drug addiction. We used a 2-D gel-based proteomics approach to examine morphine-induced temporal changes in protein expression and/or PTM in the nucleus accumbens (NAc) of morphine-sensitized rats. Rats were pretreated with saline [1 mL/kg subcutaneously (s.c.)] or morphine (10 mg/kg, s.c.) once daily for 14 days and the animals were decapitated 1 day later. The NAc was extracted and proteins resolved by 2-DE. Several protein functional groups were found to be regulated in the morphine-treated group, representing cytoskeletal proteins, proteins involved in neurotransmission, enzymes involved in energy metabolism and protein degradation, and a protein that regulates translation.     

[3H]Noradrenaline Release from Brain Slices Induced by an Increase in the Intracellular Sodium Concentration: Role of Intracellular Calcium Stores

Rat brain slices, prelabeled with [3H]noradrenaline, were superfused and exposed to K+ depolarization (10-120 mM K+) or to veratrine (1-25 microM). In the absence of extracellular Ca2+ veratrine, in contrast to K+-depolarization, caused a substantial release of [3H]noradrenaline, which was completely blocked by tetrodotoxin (0.3 microM). The Ca2+ antagonist Cd2+ (50 microM), which strongly reduced K+-induced release in the presence of 1.2 mM Ca2+, did not affect release induced by veratrine in the absence of extracellular Ca2+. Ruthenium red (10 microM), known to inhibit Ca2+-entry into mitochondria, enhanced veratrine-induced [3H]noradrenaline release. Compared with K+ depolarization in the presence of 1.2 mM Ca2+, veratrine in the absence of Ca2+ caused a somewhat delayed release of [3H]noradrenaline. Further, in contrast to the fractional release of [3H]noradrenaline induced by continuous K+ depolarization in the presence of 1.2 mM Ca2+, that induced by prolonged veratrine stimulation in the absence of Ca2+ appeared to be more sustained. The data strongly suggest that veratrine-induced [3H]noradrenaline release in the absence of extracellular Ca2+ is brought about by a mobilization of Ca2+ from intracellular stores, e.g., mitochondria, subsequent to a strongly increased intracellular Na+ concentration. This provides a model for establishing the site of action of drugs that alter the stimulus-secretion coupling process in central noradrenergic nerve terminals.     

μ-opioid receptor-mediated inhibition of the release of radiolabelled noradrenaline and acetylcholine from rat amygdala slices

Presynaptic modulation by opioids of electrically-evoked neurotransmitter release from superfused rat amygdala slices prelabelled with [³H]noradrenaline (NA) and [¹⁴C]choline was examined. Both [³H]NA and [¹⁴C]acetylcholine release were strongly inhibited by morphine, the mixed δ/μ-receptor agonist [ -Ala², -Leu⁵]enkephalin (DADLE) and the highly selective μ-agonist [ -Ala², MePhe⁴, Gly-ol⁵]enkephalin (DAMGO), whereas the highly selective δ-agonist [ -Pen², -Pen⁵]enkephalin and the κ-agonist bremazocine were without effect. The inhibitory effects were potently antagonized by naloxone but not by the selective δ-receptor antagonist fentanylisothiocyanate. When the selective uptake inhibitor desipramine was used to prevent uptake of [³H]NA into noradrenergic nerve terminals, but sparing the uptake into dopaminergic nerve terminals, the electrically evoked release of tritium was strongly inhibited by bremazocine but not by DADLE or DAMGO.

The data indicate, that in the amygdala transmitter release from dopaminergic nerve fibres is inhibited only via activation of κ-receptors, whereas transmitter release from noradrenergic and cholinergic nerve fibers is subjected to inhibition by opioids via activation of μ-receptors only. Regional differences and similarities of modulation of neurotransmitter release by opioids in the rat brain are briefly discussed.     

Comparison between electrically evoked and potassium-induced 3H-noradrenaline release from rat neocortex slices: Role of calcium ions and transmitter pools

In this study ³H-noradrenaline (NA) release from rat neocortex slices evoked by electrical field-stimulation (1 Hz, 12 mA, 2 msec) was compared with that induced by K⁺-depolarization (13–30 mM K⁺) under similar experimental conditions, with a particular emphasis on the role of external Ca²⁺ and the releasable transmitter pool(s). Not only ³H-NA release evoked by electrical stimulation but also that induced by 13 mM K⁺ was almost completely blocked by 0.3 μM tetrodotoxin (TTX). Release induced by 20 mM K⁺ appeared to be less sensitive to TTX. Thus, under relatively mild stimulation conditions, the activation of sodium channels appears to be involved in ³H-NA release elicited by both stimuli.The electrically evoked ³H-NA release increased sigmoidally with the external Ca²⁺-concentration up to 1.2 mM. In contrast, ³H-NA release induced by 13–20 mM K⁺ reached a maximal value at 0.6–0.9 mM Ca²⁺ and gradually decreased at higher Ca²⁺-concentrations. The Ca²⁺-antagonist D-600 (1–30 μM) did not inhibit electrically evoked release, while K⁺-induced ³H-NA release was dose-dependently reduced. Upon repetitive K⁺-depolarization a strong depression of ³H-NA release could be demonstrated, while this phenomenon did not occur with repeated electrical stimulation. Moreover, a previous K⁺-induced (partial) depletion of ³H-NA stores did not affect the release evoked by electrical pulses and vice versa. Taken together these data are compatible with a much stronger activation of Ca²⁺-channels and a larger vesicle mobilizing capacity in case of electrical stimulation at physiological frequencies compared to sustained depolarization with moderate K⁺-concentrations.