1 The mechanism underlying 5-hydroxytryptamine (5-HT) and/or dopamine release induced by (+)-amphetamine ((+)-Amph), 3,4-methylendioxymethamphetamine (MDMA), p-chloroamphetamine (pCA) and (+)-fen¯uramine ((+)-Fen) was investigated in rat brain superfused synaptosomes preloaded with the 3 H neurotransmitters. 2 Their rank order of potency for [ 3 H]-5-HT-releasing activity was the same as for inhibition of 5-HT uptake (pCA5MDMA5(+)-Fen44(+)-Amph). Similarly, their rank order as [ 3 H]-dopamine releasers and dopamine uptake inhibitors was the same ((+)-Amph44pCA=MDMA44(+)-Fen). We also con®rmed that the release induced by these compounds was prevented by selective transporter inhibitors (indalpine or nomifensine). The inhibitory e ect of methiothepin was not due to its e ects as a transporter inhibitor or Ca 2+ -channel blocker and is unlikely to be due to its antagonist properties on 5-HT 1/2 , dopamine or any other extracellular receptor. 5 These results indicate that the release induced by these compounds is both`carrier-mediated' and Ca 2+ -dependent (possibly exocytotic-like), with the speci®c carrier allowing the amphetamines to enter the synaptosome. The Ca 2+ -dependent release is mediated by Ca 2+ -in¯ux (mainly through P-type Ca 2+ -channels), possibly triggered by the drug interacting with an unknown intracellular target, a ected by methiothepin, common to both 5-HT and dopamine synaptosomes.

Glioblastoma (GBM) is a deadly primary brain malignancy with extensive intratumoral hypoxia. Hypoxic regions of GBM contain stem-like cells and are associated with tumor growth and angiogenesis. The molecular mechanisms that regulate tumor growth in hypoxic conditions are incompletely understood. Here, we use primary human tumor biospecimens and cultures to identify GPR133 (ADGRD1), an orphan member of the adhesion family of G-protein-coupled receptors, as a critical regulator of the response to hypoxia and tumor growth in GBM. GPR133 is selectively expressed in CD133+ GBM stem cells (GSCs) and within the hypoxic areas of PPN in human biospecimens. GPR133 mRNA is transcriptionally upregulated by hypoxia in hypoxia-inducible factor 1α (Hif1α)-dependent manner. Genetic inhibition of GPR133 with short hairpin RNA reduces the prevalence of CD133+ GSCs, tumor cell proliferation and tumorsphere formation in vitro. Forskolin rescues the GPR133 knockdown phenotype, suggesting that GPR133 signaling is mediated by cAMP. Implantation of GBM cells with short hairpin RNA-mediated knockdown of GPR133 in the mouse brain markedly reduces tumor xenograft formation and increases host survival. Analysis of the TCGA data shows that GPR133 expression levels are inversely correlated with patient survival. These findings indicate that GPR133 is an important mediator of the hypoxic response in GBM and has significant protumorigenic functions. We propose that GPR133 represents a novel molecular target in GBM and possibly other malignancies where hypoxia is fundamental to pathogenesis.

The effect in rats of chronic treatment with two specific 5-HT reuptake inhibitors (SSRI) with antidepressant properties, citalopram (10 mg/kg, i.p. twice a day for 14 days, one day washout) and fluoxetine (15 mg/kg, p.o. twice a day for 21 days, 7 days washout), was evaluated on some mechanisms involved in central 5-HT neurotransmission. No adaptive modifications of brain 5-HT uptake (sites) were found by measuring functional [3H]5-HT uptake and [3H]citalopram binding in cortical and hippocampal synaptosomes, and by [3H]citalopram binding autoradiography in the raphe nuclei (5-HT cell bodies) and the ventral tegmental area (5-HT axonal pathway). Chronic treatments had no effect on presynaptic 5-HT1B autoreceptors, functionally evaluated by measuring 5-HT1B-mediated inhibition of depolarization-induced [3H]5-HT release from cortical and hippocampal synaptosomes. Chronic citalopram or fluoxetine did not significantly affect the binding of [3H]BRL-43694 to 5-HT3 receptors in the rat brain cortex. Citalopram had no effect on [125I]SB-207710 binding to 5-HT4 receptors, measured by autoradiography in the substantia nigra. Negative results, such as those reported in the present study, could be due to a number of variables including the animal species, the treatment schedule or the brain areas considered, thus explaining the differences from some previous reports of significant effects of SSRI. However, our negative data are in agreement with many other published studies, suggesting that adaptive modifications of brain 5-HT transporters, terminal 5-HT1B receptors, 5-HT3 and 5-HT4 receptors may not be a general effect induced by all SSRI.

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The effect of cracked corn grain supplementation (3.5 kg/day) during 3 weeks before the expected calving date on milk production and composition, body condition score (BCS), metabolic and hormonal profiles and length of postpartum anoestrus was evaluated in multiparous Holstein dairy cows under grazing conditions (Energy supplemented group, n = 10; Control group, n = 10). Body condition score was weekly recorded during the peripartum period, from days -21 to +35 (parturition = day 0). Non-esterified fatty acids, beta-hydroxybutyrate, cholesterol, urea, insulin, insulin-like growth factor I (IGF-I), leptin, thyroxine (T(4)) and 3,3''5-triiodothyroinine (T(3)) were weekly determined in plasma from days -21 to +35. The reinitiation of ovarian cyclicity was twice weekly determined by ovarian ultrasonography and confirmed by plasma progesterone concentrations. Cows fed energy concentrate prepartum had higher BCS during the prepartum and postpartum and produced more milk. Non-esterified fatty acids plasma concentrations were significantly higher in the energy group, while cholesterol was higher in the control group. Treated cows had higher levels of plasma insulin, IGF-I and leptin pre-calving. IGF-I, leptin and T(4) were diminished during the early postpartum period in both groups. Insulin levels were also diminished in the control group, but levels remained high in the energy-supplemented group. Treated cows ovulated sooner after parturition than controls. We conclude that Energetic supplementation prepartum in cows under grazing conditions increased milk production and reduced the reinitiation of ovarian activity, consistent with a better EB (BCS), higher prepartum levels of IGF-I, leptin and insulin, and higher insulin levels during early postpartum.

Chronic engagement of the T cell receptor mediates the induction of T lymphocyte unresponsiveness called clonal anergy. The development of such unresponsiveness has been suggested as one of the mechanisms that regulate peripheral tolerance to self‐antigens and hamper the capacity of tumor antigen‐specific T cells to eliminate cancerous cells. In the attempt to enhance the effector function of CD4+ T lymphocytes and their resistance to clonal anergy induction, we have transduced primary T cells with a retroviral vector encoding active p21ras (RasLeu61). Here we show that RasLeu61 elicited TCR‐independent activation of the Ras‐Raf‐ERK pathway and conferred primary T cells with the ability to secrete IL‐2 in response to stimulation with a Ca2+ ionophore alone, without altering antigen‐, CD3/CD28‐ and PMA/ionomycin‐driven IL‐2 secretion and T cell proliferation in vitro. However, chronic engagement of the TCR onthe surface of RasLeu61 T cells still led to an inability of the cells to produce IL‐2 upon restimulation. These results indicate that enforced p21ras functionality enhances primary T cells responses to calcium‐generated signals, but is insufficient to prevent TCR‐driven T cell unresponsiveness and suggest that additional biochemical mechanisms, independent of p21ras, negatively regulate IL‐2 production in unresponsive T cells.

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