Marija Čarman-Kržan

Monoaminergic neuronal activity up-regulates BDNF synthesis in cultured neonatal rat astrocytes.

Astrocytes as an active part of the tripartite synapse can respond to the synaptically released neurotransmitters. Because brain-derived neurotrophic factor (BDNF) is produced by astrocytes, in addition to neurons, we focused our present study on the regulatory effects of monoamines noradrenaline (NA), serotonin (5-HT), and dopamine (DA) on the synthesis of BDNF protein in rat neonatal astrocytes from specific brain regions (cortex, cerebellum). All tested neurotransmitters are able to potently and transiently increase BDNF cellular contents; their maximal effects are dose and time dependent and differ between the two brain regions. In cultured cortical astrocytes, NA (1 microM; 6 h) elevates BDNF levels by a 4-fold, 5-HT (1 microM; 4 h) by a 2.3-fold, and DA (150 microM; 4 h) by a 2.2-fold. In cerebellar astrocytes, NA (1 microM; 4 h) increases BDNF content by a 4.7-fold, 5-HT (1 microM; 4 h) by a 1.7-fold, and DA (150 microM; 4 h) by a 1.4-fold. The initial increase in the BDNF levels return to basal levels when incubation with monoamines is extended beyond 12 h (for 5-HT) or 24 h (for NA and DA). Our results confirm the involvement of monoaminergic systems in the regulation of BDNF production in astrocytes and suggest the existence of a positive reciprocal interaction between monoaminergic neuronal activity and astrocytic neurotrophic support in neuron-astrocyte crosstalk, which has a dynamic role in mediating neuronal plasticity and trophic functions in the brain.

Interleukin-1β, but not IL-1α, mediates nerve growth factor secretion from rat astrocytes via type I IL-1 receptor

In astrocytes, nerve growth factor (NGF) synthesis and secretion is stimulated by the cytokine interleukin‐1β (IL‐1β). In the present study, the role of IL‐1 receptor binding sites in the regulation of NGF release was evaluated by determining the pharmacological properties of astroglially localized IL‐1 receptors, and, by comparing the effects of both the agonists (IL‐1α and IL‐1β) and the antagonist (IL‐1ra)—members of the IL‐1 family on NGF secretion from rat neonatal cortical astrocytes in primary culture. Using receptor‐binding studies, binding of [125I] IL‐1β to cultured astrocytes was saturable and of high affinity. Mean values for the KD and Bmax were calculated to be 60.7±7.4 pM and 2.5±0.1 fmol mg‐1 protein, respectively. The binding was rapid and readily reversible. IL‐1 receptor agonists IL‐1α (Ki of 341.1 pM) and IL‐1β (Ki 59.9 pM), as well as the antagonist IL‐1ra (Ki 257.6 pM), displaced specific [125I] IL‐1β binding from cultured astrocytes in a monophasic manner. Anti‐IL‐1RI antibody completely blocked specific [125I] IL‐1β binding while anti‐IL‐1RII antibody had no inhibitory effect. Exposure of cultured astrocytes to IL‐1α and IL‐1β revealed the functional difference between the agonists in influencing NGF release. In contrast to IL‐1β (10 U/ml), which caused a 3‐fold increase in NGF secretion compared to control cells, IL‐1α by itself had no stimulatory action on NGF release. The simultaneous application of IL‐1α and IL‐1β elicited no additive response. IL‐1ra had no effect on basal NGF release but dose‐dependently inhibited the stimulatory response induced by IL‐1β. We concluded that IL‐1β‐induced NGF secretion from cultured rat cortical astrocytes is mediated by functional type I IL‐1 receptors, whereas IL‐1α and IL‐1ra, in spite of their affinity for IL‐1RI, have no effect on NGF secretion from these cells. Type II IL‐1R is not present on rat neonatal cortical astrocytes.

Regulatory role of monoamine neurotransmitters in astrocytic NT-3 synthesis

Astrocytes actively control neuronal activity and synaptic transmission and by producing various neurotrophic factors represent an important local cellular source of trophic support in the normal and diseased brain. Our present study showed the ability of astrocytes to synthesize neurotrophin‐3 (NT‐3) and the active involvement of the monoamine neurotransmitters noradrenaline, adrenaline, dopamine, and serotonin, as well as basic intracellular second messenger systems, in the regulation of NT‐3 production in neonatal rat cortical astrocytes.

Cytokine-regulated secretion of nerve growth factor from cultured rat neonatal astrocytes

Abstract Interactions involved in the regulation of nerve growth factor (NGF) release by inflammatory cytokines: interleukin-1β (IL-1β), interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α) and transforming growth factor-β1 (TGF-β1) were examined in rat neonatal cortical astrocytes in primary culture. Abstract Exposure of cultured astrocytes to IL-1β, IL-6, and TGF-β1 resulted in the stimulation of NGF secretion. Treatment of cells for 24 h with IL-1β (10 U/ml), IL-6 (5 ng/ml) and TGF-β1 (5 ng/ml) caused 3-, 1.8-, and 2.8-fold increase in NGF secretion as compared to the control cells. In contrast, TNF-α (30 ng/ml) by itself had no stimulatory action on NGF release whereas co-stimulation of astrocytes with IL-1β and TNF-α showed a synergistic interaction. Co-treatment of astrocytes with IL-1β and TGF-β1 increased NGF secretion in an additive way, whereas simultaneous application of IL-1β and IL-6 resulted in the inhibitory effect on NGF secretion. Our results suggest that interactions between cytokines used cause the stimulation of NGF secretion through different regulatory mechanisms.

Specific binding of [3H]mepyramine to histamine H1-receptors in vascular smooth muscle membranes.

The antagonist-sensitive binding of [3H]mepyramine to beef aortic membranes was as expected for binding to histamine H1-receptors. [3H]mepyramine binds rapidly and in saturable fashion to the specific receptor sites, specific binding reaching equilibrium in 3 min at 37°CScatchards analysis of the binding data gave a dissociation constant of 3.0 nM for the radioligand-receptor complex and maximal number of binding sites: 31 fmol/mg protein. In the competition studies histamine H1-antagonists are more potent inhibitors of radioligand binding than H2-antagonist. They inhibit [3H]mepyramine binding in the following order: mepyramine >triprolidine<promethazine ≫ cimetidine. Binding data are in correlation with the previous pharmacological studies.

The effect of group selective reagentsN-ethylmaleimide and dithiothreitol on histamine H1-receptor binding sites in the vascular smooth muscle membranes

The chemical nature of the histamine H1-receptors of beef aortic membranes has been elucidated by introducing two group selective reagents in the [3H]-mepyramine binding studies: dithiothreitol (DTT), a protein-disulphide group reducing reagent, andN-ethylmaleimide (NEM), a proteinthiol group alkylating agent.In the binding experiments, NEM independently inhibits [3H]-mepyramine binding. The inhibition is time and concentration dependent. DTT on the other hand potentiates the binding of the radioligand to its receptor and changes the affinity of histamine in competing for [3H]-mepyramine binding site. In the DTT-pretreated membranes (100 μM), histamine shows a higher affinity for [3H]-mepyramine binding (Ki 0.35 μM) than in the untreated membranes (Ki 3.7 μM). Comparison of the pharmacological studies on the DTT-treated rabbit aortic strips and above binding studies, revealed a good correlation between the changes in the affinity of histamine for its receptor, when DTT was present. The results suggest an important role of the S-S and SH groups in the function of aortic histamine H1-receptor.

Effects of histamine and IL-1β on PKC-stimulated nerve growth factor secretion from glial cells

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Comparison of histamine and serotonin release from rat peritoneal mast cells induced by nerve growth factor and compound 48/80.

Nerve growth factor (NGF) is a polypeptide essential for the development and function of neurones in the CNS and PNS. It elicits several biological effects on neuronal and nonneuronal tissues [1]. More recently NGF has been shown to induce histamine release from rat peritoneal mast cells by interacting with the specific membrane NGF receptor [2]. Our previous studies showed that histamine and serotonin release induced by compound 48/80 were mediated by the interaction with a mast cell membrane binding site for basic secretagogues [3]. Since histamine release induced by compound 48/80 is more pronounced than serotonin release, it has been suggested that mast cells may release histamine and serotonin to different extents [4]. After secretion, mast cells are capable of taking up serotonin. The uptake occurs at the same concentration as the release [5, 6], whereas histamine is taken up at higher concentrations. Therefore, the apparent difference between the amine secretion may be also due to differential reuptake of the amines after the secretion. On the basis of differences between the secretion of the amines, it was of interest to compare the release of both amines induced by two secretagogues (NGF and compound 48/80), having different mechanisms of action.

Characterization of vascular histamine H1-receptors: Multiple affinity states of aortic histamine H1-receptor

We have shown that [3H]mepyramine labels histamine H1-receptor-binding sites in bovine aortic membranes. Further characterization of H1-receptors in this tissue was done by the interaction of an unlabelled histamine receptor agonist or antagonist, with the radioantagonist [3H]mepyramine-binding sites. The competition-binding assays have uncovered differences in the characteristics of the agonist/receptor interaction not shared by antagonists. Agonists interact in the heterogeneous manner with the radioantagonist-labelled sites, showing shallow competition curves with thenH 0.50–0.72, whereas antagonists were devoid of this effect (steeper slopes of the inhibition curvesnH≈1). The results suggest the presence in this tissue of multiple affinity states of histamine H1-receptor, differentiated by high and low affinity for agonists and the same affinity for antagonists.

Characterization of coexistent histamine H1- and H2-receptor binding sites in the purified guinea pig myocardial membranes from ventricles

In the present work, we identified and characterised histamine H1-and H2-receptors in highly purified myocardial membranes isolated from female guinea pig ventricles. We determined the binding parameters for the interactions of3H-mepyramine with the histamine H1-receptor binding site and3H-tiotidine with the histamine H2-receptor binding site. Binding of both ligands in our study was saturable, reversible and of high affinity. Scatchards analysis of the specific3H-mepyramine binding revealed the existence of high and low affinity binding sites with apparent KD values of 0.4 nM and 4.5 nM, respectively. The density of binding sites (Bmax) was 100 fmol/mg protein for the high and 466 fmol/mg protein for the low affinity binding site.3H-tiotidine binds to a single population of binding sites with a KD of 1.0 nM and a Bmax of 27 fmol/mg protein. These data suggest that both histamine H1-and H2-receptors coexist in the guinea pig myocardium with a significantly higher prevalence of the histamine H1-receptor population.