Ca2؉ elevations in Chinese hamster ovary cells stably expressing OX 1 receptors were measured using fluorescent Ca 2؉ indicators fura-2 and fluo-3. Stimulation with orexin-A led to pronounced Ca 2؉ elevations with an EC 50 around 1 nM. When the extracellular [Ca 2؉ ] was reduced to a submicromolar concentration, the EC 50 was increased 100-fold. Similarly, the inositol 1,4,5-trisphosphate production in the presence of 1 mM external Ca 2؉ was about 2 orders of magnitude more sensitive to orexin-A stimulation than in low extracellular Ca influx and (ii) a direct stimulation of phospholipase C, and that these two responses converge at the level of phospholipase C where the former markedly enhances the potency of the latter.The recently described hypothalamic peptides called orexins (1) or hypocretins (2) mediate their effects through G proteincoupled receptors called OX 1 and OX 2 receptors (1). The peptides and their receptors are widespread in the hypothalamus, cortex, and brainstem (2-5). The orexin/hypocretin peptides are encoded by a single mRNA giving rise to a 33-residue orexin-A peptide containing disulfide bridges and a linear 28-residue orexin-B (1). Orexin-A has a 10 -100-fold higher affinity and potency for OX 1 receptor as compared with orexin-B, whereas no preference is displayed by the OX 2 receptor (1). The orexins cause robust increases in intracellular Ca 2ϩ both in neurons cultured from rat medial and lateral hypothalamus (6) and spinal cord (7), and when studied using recombinant receptors (1). This has led to the suggestion that the receptors are coupled to the G q family G proteins. Interestingly, the response in neurons is partially dependent on extracellular Ca 2ϩ , which may suggest that the receptors are connected to a Ca 2ϩ influx pathway in neurons (6). Several different pathways for receptor-stimulated Ca 2ϩ entry have been suggested based on functional studies with other G protein-coupled receptors. Suggested pathways include store-operated Ca 2ϩ channels, second messenger-operated channels, as well as Ca 2ϩ -activated Ca 2ϩ channels (reviewed in Refs. 8 and 9). The aim of this study was to examine in detail the Ca 2ϩ mobilizing actions of orexins on recombinant OX 1 receptors expressed in CHO 1 -K1 cells. The results reveal the presence of a novel amplification mechanism at the level of phospholipase C that is dependent on activation of Ca 2ϩ influx pathway upstream of phospholipase C. EXPERIMENTAL PROCEDURESCell Cultures-To prepare the CHO-hOX 1 -C1 cells used in this study CHO-K1 cells were transfected with a bicistronic vector containing the coding sequence of human OX 1 receptor as described previously for chemokine receptors (10). Neomycin resistant clones were then isolated by limited dilution. They were grown in nutrient mixture (Ham's F-12) medium (Life Technologies, Inc., Paisley, United Kingdom) supplemented with 100 units/ml penicillin G (Sigma), 80 units/ml streptomycin (Sigma), 400 g/ml Geneticin (G418; Life Technologies, Inc.) and 10% (v/v) fetal calf serum (Life Te...

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Serotonin (5-HT) system has a significant role in anxiety- and depression-related states and may be influenced by brain-derived neurotrophic factor (BDNF). This study examined extracellular 5-HT levels and expression of BDNF in rats with persistently low or high levels of exploratory activity (LE and HE, respectively). Baseline extracellular levels of 5-HT as assessed by in vivo microdialysis in conscious animals were similar in both groups in medial prefrontal cortex (PFC) and dentate gyrus (DG). No differences were found in parachloroamphetamine-induced 5-HT release in either region. However, LE animals had significantly higher levels of 5-HT transporter (5-HTT) binding in PFC and a larger increase in extracellular 5-HT levels after administration of citalopram (1 μM) into this area by retrograde dialysis. No difference in 5-HTT levels was found in hippocampus, while perfusion with citalopram was accompanied by a greater increase in extracellular 5-HT in the HE group in this brain region. LE-rats had higher levels of BDNF mRNA in the PFC but not hippocampus. In contrast, levels of nerve growth factor mRNA were similar in these brain regions of LE- and HE-rats. The differential regulation of 5-HT-ergic system in LE- and HE-rats in PFC and hippocampus may form the basis for their distinct anxiety-related behaviours.

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Novel anticancer compounds and their precision delivery systems are actively developed to create potent and well-tolerated anticancer therapeutics.H ere,w er eport the synthesis of an ovel anthracycline,U torubicin (UTO), and its preclinical development as an anticancer payload for nanocarriers.F ree UTOw as significantly more toxic to cultured tumor cell lines than the clinically used anthracycline,d oxorubicin. Nanoformulated UTO, encapsulated in polymeric nanovesicles (polymersomes,P S), reduced the viability of cultured malignant cells and this effect was potentiated by functionalization with at umor-penetrating peptide (TPP). Systemic peptide-guided PS showed preferential accumulation in triple-negative breast tumor xenografts implanted in mice. At the same systemic UTOd ose,t he highest UTOa ccumulation in tumor tissue was seen for the TPP-targeted PS, followed by nontargeted PS,a nd free doxorubicin. Our study suggests potential applications for UTOi nt he treatment of malignant diseases and encourages further preclinical and clinical studies on UTOasananocarrier payload for precision cancer therapy.