The Differentiation of Skin Mesenchymal Stem Cells Towards a Schwann Cell Phenotype: Impact of Sigma-1 Receptor Activation.

Neural crest stem cells (NCSCs) are the source of mature Schwann cells in the peripheral nervous system (PNS). The NCSC population resides in the bulge of hair follicles and in the dermis. Recently, it was shown that 2-3% of the human dermis mesenchymal stem cell (MSC) population expresses the NCSC marker CD271, thus enabling the use of skin MSCs for studying Schwann cell differentiation in vitro. The aims of this study were to establish a protocol for human skin MSC differentiation towards Schwann cell-like cells (SC-lcs) and to analyse the expression of sigma-1 receptor (S1R) in SC-lcs. The impact of S1R ligands, namely the selective agonist PRE-084, the positive allosteric modulator E1R and the selective antagonist NE-100, on Schwann cell differentiation was assessed. The expression of the neuron-specific genes Tubulin-ßIII and Integrin-6α, the Schwann cell-specific gene S100b, MBP and the NCSC-specific genes p75NTR, Sox10, Notch1, Integrin-4α, Ap2α and Pax6 was analysed in MSCs and SC-lcs by real-time RT-PCR. BDNF secretion was evaluated by ELISA. The effect of S1R ligands on SC-lc differentiation was measured using BDNF ELISA and MBP flow cytometry. After MSC differentiation, NCSC markers p75NTR and Integrin-4α were downregulated 3.5-fold and 2-fold, respectively. To the contrary, MBP and S100b were significantly upregulated in SC-lcs. S1R ligands showed a tendency to increase the secretion of BDNF by the SC-lc population. PRE-084 and E1R increased MBP expression in the SC-lc population, whereas 3 µM NE-100 inhibited MBP expression in SC-lcs. In conclusion, our data demonstrate that S1R plays an important role in skin MSC differentiation towards myelinating Schwann cells.

The cognition-enhancing activity of E1R, a novel positive allosteric modulator of sigma-1 receptors.

BACKGROUND AND PURPOSE: Here, we describe the in vitro and in vivo effects of (4R,5S)-2-(5-methyl-2-oxo-4-phenyl-pyrrolidin-1-yl)-acetamide (E1R), a novel positive allosteric modulator of sigma-1 receptors. EXPERIMENTAL APPROACH: E1R was tested for sigma receptor binding activity in a [³H](+)-pentazocine assay, in bradykinin (BK)-induced intracellular Ca²âº concentration ([Ca²âº](i)) assays and in an electrically stimulated rat vas deferens model. E1R's effects on cognitive function were tested using passive avoidance (PA) and Y-maze tests in mice. A selective sigma-1 receptor antagonist (NE-100), was used to study the involvement of the sigma-1 receptor in the effects of E1R. The open-field test was used to detect the effects of E1R on locomotion. KEY RESULTS: Pretreatment with E1R enhanced the selective sigma-1 receptor agonist PRE-084's stimulating effect during a model study employing electrically stimulated rat vasa deferentia and an assay measuring the BK-induced [Ca²âº](i) increase. Pretreatment with E1R facilitated PA retention in a dose-related manner. Furthermore, E1R alleviated the scopolamine-induced cognitive impairment during the PA and Y-maze tests in mice. The in vivo and in vitro effects of E1R were blocked by treatment with the selective sigma-1 receptor antagonist NE-100. E1R did not affect locomotor activity. CONCLUSION AND IMPLICATIONS: E1R is a novel 4,5-disubstituted derivative of piracetam that enhances cognition and demonstrates efficacy against scopolamine-induced cholinergic dysfunction in mice. These effects are attributed to its positive modulatory action on the sigma-1 receptor and this activity may be relevant when developing new drugs for treating cognitive symptoms related to neurodegenerative diseases.

EPR investigation of in vivo inhibitory effect of guanidine compounds on nitric oxide production in rat tissues.

The aim of the present study was to evaluate in vivo effects on NO production of pharmacologically widely used, commercially available NOS inhibitors, structurally related to guanidine. We compared the NO inhibitory potency and selectivity of L-NAME, aminoguanidine and guanabenz in tissues of normal and LPS-stimulated rats using ex vivo EPR measurements of the NO radical in its complex with dithiocarbamate-Fe(II). The tissues studied were the brain cortex, kidney, liver, heart and testis. Differential inhibitory effects were seen for L-NAME, aminoguanidine and guanabenz when applied during basal or LPS-stimulated conditions. Aminoguanidine exerted inhibition of NO only after stimulation with LPS. Guanabenz had little effect on NO in liver, kidney, testis and heart under normal conditions, while it reduced the basal NO in brain cortex. After stimulation with LPS guanabenz afforded a partial inhibition of the NO formation in all tissues studied. L-NAME was a potent inhibitor of NO synthesis in all tested tissues, both during basal and LPS stimulated conditions. Our results show that compounds containing a guanidine moiety might possess different NOS inhibitory profiles in vivo.

Increased synthesis of nitric oxide in rat brain cortex due to halogenated volatile anesthetics confirmed by EPR spectroscopy.

BACKGROUND: Halogenated volatile anesthetics (HVAs) are considered to be inhibitors of nitric oxide synthase (NOS). On other hand, NO mediates the vasodilation produced by HVAs. Thus, both increase and decrease of NO concentration in brain tissues are possible during anesthesia. Previously, we have observed an increase of NO content in rat brain cortex under halothane anesthesia. The goal of this study was to determine whether the observed phenomenon was general for this anesthetic group, if it was specific for brain cortex, and if the NO increase was due changes in NOS activity. METHODS: NO scavengers were injected to adult rats 30 min prior to anesthesia. Rats were anesthetized by inhalation of an O2 mixture with volatile anesthetics (1.5% for halothane; 1% for isoflurane, 2% for sevoflurane). After 30 min of anesthesia, rats were decapitated and brain cortex, cerebellum, liver, heart, kidneys and testes were dissected, frozen in liquid nitrogen and subjected to EPR spectroscopy. Nitric oxide content was determined quantitatively based on the intensity of the NO-Fe-DETC complex spectrum and its comparison with the calibration curve. RESULTS: In rats anesthetized with HVAs, we observed a greater than twofold increase of NO content in brain cortex as compared to the nonanesthetized animals. No significant changes were detected in other organs. The NOS inhibitor N(omega)-nitro-L-arginine abolished the increase of NO content in brain produced by volatile anesthetics. CONCLUSION: The action of volatile anesthetics is coupled with an increase of NO content in the cortex dependent on NOS activity.