Effects of Side Chain and Peptide Bond Modifications on the Targeting Properties of Stabilized Minigastrin Analogs.

Different attempts have been made in the past two decades to develop radiolabeled peptide conjugates with enhanced pharmacokinetic properties in order to improve the application for tumor imaging and peptide receptor radionuclide therapy (PRRT), which targets the cholecystokinin-2 receptor (CCK2R). In this paper, the influence of different side chain and peptide bond modifications has been explored for the minigastrin analog DOTA-DGlu-Ala-Tyr-Gly-Trp-(N-Me)Nle-Asp-1Nal-NH2 (DOTA-MGS5). Based on this lead structure, five new derivatives were synthesized for radiolabeling with trivalent radiometals. Different chemical and biological properties of the new derivatives were analyzed. Receptor interaction of the peptide derivatives and cell internalization of the radiolabeled peptides were studied in A431-CCK2R cells. The stability of the radiolabeled peptides in vivo was investigated using BALB/c mice. Tumor targeting of all 111In-labeled peptide conjugates, and of a selected compound radiolabeled with gallium-68 and lutetium-177, was evaluated in BALB/c nude mice xenografted with A431-CCK2R and A431-mock cells. All 111In-labeled conjugates, except [111In]In-DOTA-[Phe8]MGS5, showed a high resistance against enzymatic degradation. A high receptor affinity with IC50 values in the low nanomolar range was confirmed for most of the peptide derivatives. The specific cell internalization over time was 35.3-47.3% for all radiopeptides 4 h after incubation. Only [111In]In-DOTA-MGS5[NHCH3] exhibited a lower cell internalization of 6.6 ± 2.8%. An overall improved resistance against enzymatic degradation was confirmed in vivo. Of the radiopeptides studied, [111In]In-DOTA-[(N-Me)1Nal8]MGS5 showed the most promising targeting properties, with significantly increased accumulation of radioactivity in A431-CCK2R xenografts (48.1 ± 9.2% IA/g) and reduced accumulation of radioactivity in stomach (4.2 ± 0.5% IA/g). However, in comparison with DOTA-MGS5, a higher influence on the targeting properties was observed for the change of radiometal, resulting in a tumor uptake of 15.67 ± 2.21% IA/g for [68Ga]Ga-DOTA-[(N-Me)1Nal8]MGS5 and 35.13 ± 6.32% IA/g for [177Lu]Lu-DOTA-[(N-Me)1Nal8]MGS5.

Chemotactic cell migration: the core autophagy protein ATG9A is at the leading edge.

Accumulating data indicate that several components of the macroautophagy/autophagy machinery mediate additional functions, which do not depend on autophagosome biogenesis or lysosomal cargo degradation. In this context, we found that the core autophagy protein ATG9A participates in the chemotactic movement of several cell lines, including highly invasive glioblastoma cells. Accordingly, ATG9A-depleted cells are unable to form large and persistent leading-edge protrusions. By the design of an ATG9A-pHluorin construct and TIRF imaging, we established that ATG9A-positive vesicles are targeted toward the migration front, where their exocytosis is synchronized with protrusive activity. We finally demonstrated that ATG9A, through its interaction with clathrin adaptor complexes, controls the delivery of ITGB1 (integrin subunit beta 1) to the migration front and normal adhesion dynamics. Together, our work indicates that ATG9A protein has a wider role than anticipated and constitutes a critical component of vesicular trafficking allowing the expansion of cell protrusions and their anchorage to the extracellular matrix.

The core autophagy protein ATG9A controls dynamics of cell protrusions and directed migration.

Chemotactic migration is a fundamental cellular behavior relying on the coordinated flux of lipids and cargo proteins toward the leading edge. We found here that the core autophagy protein ATG9A plays a critical role in the chemotactic migration of several human cell lines, including highly invasive glioma cells. Depletion of ATG9A protein altered the formation of large and persistent filamentous actin (F-actin)-rich lamellipodia that normally drive directional migration. Using live-cell TIRF microscopy, we demonstrated that ATG9A-positive vesicles are targeted toward the migration front of polarized cells, where their exocytosis correlates with protrusive activity. Finally, we found that ATG9A was critical for efficient delivery of ß1 integrin to the leading edge and normal adhesion dynamics. Collectively, our data uncover a new function for ATG9A protein and indicate that ATG9A-positive vesicles are mobilized during chemotactic stimulation to facilitate expansion of the lamellipodium and its anchorage to the extracellular matrix.

Targeting the Urotensin II/UT G Protein-Coupled Receptor to Counteract Angiogenesis and Mesenchymal Hypoxia/Necrosis in Glioblastoma.

Glioblastomas (GBMs) are the most common primary brain tumors characterized by strong invasiveness and angiogenesis. GBM cells and microenvironment secrete angiogenic factors and also express chemoattractant G protein-coupled receptors (GPCRs) to their advantage. We investigated the role of the vasoactive peptide urotensin II (UII) and its receptor UT on GBM angiogenesis and tested potential ligand/therapeutic options based on this system. On glioma patient samples, the expression of UII and UT increased with the grade with marked expression in the vascular and peri-necrotic mesenchymal hypoxic areas being correlated with vascular density. In vitro human UII stimulated human endothelial HUV-EC-C and hCMEC/D3 cell motility and tubulogenesis. In mouse-transplanted Matrigel sponges, mouse (mUII) and human UII markedly stimulated invasion by macrophages, endothelial, and smooth muscle cells. In U87 GBM xenografts expressing UII and UT in the glial and vascular compartments, UII accelerated tumor development, favored hypoxia and necrosis associated with increased proliferation (Ki67), and induced metalloproteinase (MMP)-2 and -9 expression in Nude mice. UII also promoted a "tortuous" vascular collagen-IV expressing network and integrin expression mainly in the vascular compartment. GBM angiogenesis and integrin αvß3 were confirmed by in vivo 99mTc-RGD tracer imaging and tumoral capture in the non-necrotic area of U87 xenografts in Nude mice. Peptide analogs of UII and UT antagonist were also tested as potential tumor repressor. Urotensin II-related peptide URP inhibited angiogenesis in vitro and failed to attract vascular and inflammatory components in Matrigel in vivo. Interestingly, the UT antagonist/biased ligand urantide and the non-peptide UT antagonist palosuran prevented UII-induced tubulogenesis in vitro and significantly delayed tumor growth in vivo. Urantide drastically prevented endogenous and UII-induced GBM angiogenesis, MMP, and integrin activations, associated with GBM tumoral growth. These findings show that UII induces GBM aggressiveness with necrosis and angiogenesis through integrin activation, a mesenchymal behavior that can be targeted by UT biased ligands/antagonists.

Stabilization Strategies for Linear Minigastrin Analogues: Further Improvements via the Inclusion of Proline into the Peptide Sequence.

Minigastrin (MG) analogues, known for their high potential to target cholecystokinin-2 receptor (CCK2R) expressing tumors, have limited clinical applicability due to low enzymatic stability. By introducing site-specific substitutions within the C-terminal receptor-binding sequence, reduced metabolization and improved tumor targeting can be achieved. In this work, the influence of additional modification within the N-terminal sequence has been explored. Three novel 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated CCK2R ligands with proline substitution at different positions were synthesized. Substitution did not affect CCK2R affinity, and the conjugates labeled with indium-111 and lutetium-177 showed a high enzymatic stability in different incubation media as well as in vivo (57-79% intact radiopeptide in blood of BALB/c mice at 1 h p.i.) combined with enhanced tumor uptake (29-46% IA/g at 4 h in xenografted BALB/c nude mice). The inclusion of Pro contributes significantly to the development of CCK2R ligands with optimal targeting properties for application in targeted radiotherapy.

Development of Novel 111-In-Labelled DOTA Urotensin II Analogues for Targeting the UT Receptor Overexpressed in Solid Tumours.

Overexpression of G protein-coupled receptors (GPCRs) in tumours is widely used to develop GPCR-targeting radioligands for solid tumour imaging in the context of diagnosis and even treatment. The human vasoactive neuropeptide urotensin II (hUII), which shares structural analogies with somatostatin, interacts with a single high affinity GPCR named UT. High expression of UT has been reported in several types of human solid tumours from lung, gut, prostate, or breast, suggesting that UT is a valuable novel target to design radiolabelled hUII analogues for cancer diagnosis. In this study, two original urotensinergic analogues were first conjugated to a DOTA chelator via an aminohexanoic acid (Ahx) hydrocarbon linker and then -hUII and DOTA-urantide, complexed to the radioactive metal indium isotope to successfully lead to radiolabelled DOTA-Ahx-hUII and DOTA-Ahx-urantide. The 111In-DOTA-hUII in human plasma revealed that only 30% of the radioligand was degraded after a 3-h period. DOTA-hUII and DOTA-urantide exhibited similar binding affinities as native peptides and relayed calcium mobilization in HEK293 cells expressing recombinant human UT. DOTA-hUII, not DOTA-urantide, was able to promote UT internalization in UT-expressing HEK293 cells, thus indicating that radiolabelled 111In-DOTA-hUII would allow sufficient retention of radioactivity within tumour cells or radiolabelled DOTA-urantide may lead to a persistent binding on UT at the plasma membrane. The potential of these radioligands as candidates to target UT was investigated in adenocarcinoma. We showed that hUII stimulated the migration and proliferation of both human lung A549 and colorectal DLD-1 adenocarcinoma cell lines endogenously expressing UT. In vivo intravenous injection of 111In-DOTA-hUII in C57BL/6 mice revealed modest organ signals, with important retention in kidney. 111In-DOTA-hUII or 111In-DOTA-urantide were also injected in nude mice bearing heterotopic xenografts of lung A549 cells or colorectal DLD-1 cells both expressing UT. The observed significant renal uptake and low tumour/muscle ratio (around 2.5) suggest fast tracer clearance from the organism. Together, DOTA-hUII and DOTA-urantide were successfully radiolabelled with 111Indium, the first one functioning as a UT agonist and the second one as a UT-biased ligand/antagonist. To allow tumour-specific targeting and prolong body distribution in preclinical models bearing some solid tumours, these radiolabelled urotensinergic analogues should be optimized for being used as potential molecular tools for diagnosis imaging or even treatment tools.

Urantide Improves Cardiac Function, Modulates Systemic Cytokine Response, and Increases Survival in A Murine Model of Endotoxic Shock.

INTRODUCTION: Urotensin II is a potent vasoactive peptide activating the the G protein-coupled urotensin II receptor UT, and is involved in systemic inflammation and cardiovascular functions. The aim of our work was to study the impact of the UT antagonist urantide on survival, systemic inflammation, and cardiac function during endotoxic shock. METHODS: C57Bl/6 mice were intraperitoneally injected with lipopolysaccharide (LPS) and then randomized to be injected either by urantide or NaCl 0.9% 3, 6, and 9 h (H3, H6, H9) after LPS. The effect of urantide on the survival rate, the levels of cytokines in plasma at H6, H9, H12, the expression level of nuclear factor-kappa B (NF-κB-p65) in liver and kidney (at H12), and the cardiac function by trans-thoracic echocardiography from H0 to H9 was evaluated. RESULTS: Urantide treatment improved survival (88.9% vs. 30% on day 6, P < 0.05). This was associated with changes in cytokine expression: a decrease in IL-6 (2,485 [2,280-2,751] pg/mL vs. 3,330 [3,119-3,680] pg/mL, P < 0.01) at H6, in IL-3 (1.0 [0.40-2.0] pg/mL vs. 5.8 [3.0-7.7] pg/mL, P < 0.01), and IL-1ß (651 [491-1,135] pg/mL vs. 1,601 [906-3,010] pg/mL, P < 0.05) at H12 after LPS administration. Urantide decreased the proportion of cytosolic NF-κB-p65 in liver (1.3 [0.9-1.9] vs. 3.2 [2.3-4], P < 0.01) and kidney (0.3 [0.3-0.4] vs. 0.6 [0.5-1.1], P < 0.01). Urantide improved cardiac function (left ventricular fractional shortening: 24.8 [21.5-38.9] vs. 12.0 [8.7-17.6] %, P < 0.01 and cardiac output: 30.3 [25.9-39.8] vs. 15.1 [13.0-16.9] mL/min, P < 0.0001). CONCLUSION: These results show a beneficial curative role of UT antagonism on cytokine response (especially IL-3), cardiac dysfunction, and survival during endotoxic shock in mice, highlighting a potential new therapeutic target for septic patients.

Diverted Natural Lossen-type Rearrangement for Bioconjugation through in Situ Myrosinase-Triggered Isothiocyanate Synthesis.

Fluorescein isothiocyanate (FITC) is one of the most extensively used fluorescent probes for the labeling of biomolecules. The isothiocyanate function reacts with lysine residues of proteins to provide a chemically stable thiourea linkage without releasing any byproduct. However, diversification of isothiocyanate-based reagents is still hampered by the lack of mild conditions to generate isothiocyanate chemical functions, as well as by their poor stability and limited solutions available to increase water solubility, restricting the use of isothiocyanate labeling to highly water-soluble fluorophores. Inspired by plant biological processes, we report a safe and biocompatible myrosinase-assisted in situ formation of isothiocyanate conjugates from a highly water-soluble and stable glucosinolate precursor. This method was applied for the fluorescence labeling of a plasmatic protein and fluorescence imaging of living cells.

The G Protein-Coupled Receptor UT of the Neuropeptide Urotensin II Displays Structural and Functional Chemokine Features.

The urotensinergic system was previously considered as being linked to numerous physiopathological states, including atherosclerosis, heart failure, hypertension, pre-eclampsia, diabetes, renal disease, as well as brain vascular lesions. Thus, it turns out that the actions of the urotensin II (UII)/G protein-coupled receptor UT system in animal models are currently not predictive enough in regard to their effects in human clinical trials and that UII analogs, established to target UT, were not as beneficial as expected in pathological situations. Thus, many questions remain regarding the overall signaling profiles of UT leading to complex involvement in cardiovascular and inflammatory responses as well as cancer. We address the potential UT chemotactic structural and functional definition under an evolutionary angle, by the existence of a common conserved structural feature among chemokine receptorsopioïdergic receptors and UT, i.e., a specific proline position in the transmembrane domain-2 TM2 (P2.58) likely responsible for a kink helical structure that would play a key role in chemokine functions. Even if the last decade was devoted to the elucidation of the cardiovascular control by the urotensinergic system, we also attempt here to discuss the role of UII on inflammation and migration, likely providing a peptide chemokine status for UII. Indeed, our recent work established that activation of UT by a gradient concentration of UII recruits Gαi/o and Gα13 couplings in a spatiotemporal way, controlling key signaling events leading to chemotaxis. We think that this new vision of the urotensinergic system should help considering UT as a chemotactic therapeutic target in pathological situations involving cell chemoattraction.

The Autophagy Machinery: A New Player in Chemotactic Cell Migration.

Autophagy is a highly conserved self-degradative process that plays a key role in diverse cellular processes such as stress response or differentiation. A growing body of work highlights the direct involvement of autophagy in cell migration and cancer metastasis. Specifically, autophagy has been shown to be involved in modulating cell adhesion dynamics as well as epithelial-to-mesenchymal transition. After providing a general overview of the mechanisms controlling autophagosome biogenesis and cell migration, we discuss how chemotactic G protein-coupled receptors, through the repression of autophagy, may orchestrate membrane trafficking and compartmentation of specific proteins at the cell front in order to support the critical steps of directional migration.

Chemotactic G protein-coupled receptors control cell migration by repressing autophagosome biogenesis.

Chemotactic migration is a fundamental behavior of cells and its regulation is particularly relevant in physiological processes such as organogenesis and angiogenesis, as well as in pathological processes such as tumor metastasis. The majority of chemotactic stimuli activate cell surface receptors that belong to the G protein-coupled receptor (GPCR) superfamily. Although the autophagy machinery has been shown to play a role in cell migration, its mode of regulation by chemotactic GPCRs remains largely unexplored. We found that ligand-induced activation of 2 chemotactic GPCRs, the chemokine receptor CXCR4 and the urotensin 2 receptor UTS2R, triggers a marked reduction in the biogenesis of autophagosomes, in both HEK-293 and U87 glioblastoma cells. Chemotactic GPCRs exert their anti-autophagic effects through the activation of CAPNs, which prevent the formation of pre-autophagosomal vesicles from the plasma membrane. We further demonstrated that CXCR4- or UTS2R-induced inhibition of autophagy favors the formation of adhesion complexes to the extracellular matrix and is required for chemotactic migration. Altogether, our data reveal a new link between GPCR signaling and the autophagy machinery, and may help to envisage therapeutic strategies in pathological processes such as cancer cell invasion.

Evaluation of the impact of the cancer therapy everolimus on the central nervous system in mice.

Cancer and treatments may induce cognitive impairments in cancer patients, and the causal link between chemotherapy and cognitive dysfunctions was recently validated in animal models. New cancer targeted therapies have become widely used, and their impact on brain functions and quality of life needs to be explored. We evaluated the impact of everolimus, an anticancer agent targeting the mTOR pathway, on cognitive functions, cerebral metabolism, and hippocampal cell proliferation/vascular density in mice. Adult mice received everolimus daily for 2 weeks, and behavioral tests were performed from 1 week after the last treatment. Everolimus-treated mice displayed a marked reduction in weight gain from the last day of the treatment period. Ex vivo analysis showed altered cytochrome oxidase activity in selective cerebral regions involved in energy balance, food intake, reward, learning and memory modulation, sleep/wake cycle regulation, and arousal. Like chemotherapy, everolimus did not alter emotional reactivity, learning and memory performances, but in contrast to chemotherapy, did not affect behavioral flexibility or reactivity to novelty. In vivo hippocampal neural cell proliferation and vascular density were also unchanged after everolimus treatments. In conclusion, two weeks daily everolimus treatment at the clinical dose did not evoke alteration of cognitive performances evaluated in hippocampal- and prefrontal cortex-dependent tasks that would persist at one to four weeks after the end of the treatment completion. However, acute everolimus treatment caused selective CO modifications without altering the mTOR effector P70S6 kinase in cerebral regions involved in feeding behavior and/or the sleep/wake cycle, at least in part under control of the solitary nucleus and the parasubthalamic region of the hypothalamus. Thus, this area may represent a key target for everolimus-mediating peripheral modifications, which has been previously associated with symptoms such as weight loss and fatigue.

Increased plasma levels of endozepines, endogenous ligands of benzodiazepine receptors, during systemic inflammation: a prospective observational study.

INTRODUCTION: Recent work has shown that benzodiazepines interact with the immune system and exhibit anti-inflammatory effects. By using in vitro models, researchers in several studies have shown that the peptidergic endogenous ligands of benzodiazepine receptors, named endozepines, are involved in the immune response. All endozepines identified so far derive from diazepam-binding inhibitor (DBI), which generates several biologically active fragments. The aim of the present study was to measure plasma levels of DBI-like immunoreactivity (DBI-LI) in a rat model of sepsis and in patients with systemic inflammation from septic or non-septic origin. METHODS: Cecal ligation and puncture (CLP) or sham surgery was performed in rats. Blood samples were taken from animals, patients hospitalized for digestive surgery with inflammatory diseases, and healthy volunteers. Measurements of plasma DBI-related peptides were carried out by radioimmunoassay in animal and human samples. RESULTS: In the rats, CLP provoked an increase of plasma DBI-LI (+37%) 6 hours postsurgery. In humans, DBI-LI levels were significantly higher in the systemic inflammation group than in the healthy volunteer group (48.6 (32.7 to 77.7) pg/ml versus 11.1 (5.9 to 35.3) pg/ml, P < .001). We found a positive correlation between endozepine levels and Acute Physiology and Chronic Health Evaluation II score (r s = 0.33 (0.026 to 0.58), P < 0.05) and tumor necrosis factor α levels (r s = 0.43 (0.14 to 0.65), P < 0.01). The area under the receiver operating characteristic curve for endozepines was 0.842 (95% CI (0.717 to 0.966), P < 0.0001) for discriminating patients with inflammation from healthy volunteers. CONCLUSIONS: Endozepines might be involved in the inflammatory response in patients with systemic inflammation.

Impact of meriolins, a new class of cyclin-dependent kinase inhibitors, on malignant glioma proliferation and neo-angiogenesis.

BACKGROUND: Glioblastomas are the most frequent and most aggressive primary brain tumors in adults. The median overall survival is limited to a few months despite surgery, radiotherapy, and chemotherapy. It is now clearly established that hyperactivity of cyclin-dependent kinases (CDKs) is one of the processes underlying hyperproliferation and tumoral growth. The marine natural products meridianins and variolins, characterized as CDK inhibitors, display a kinase-inhibitory activity associated with cytotoxic effects. In order to improve selectivity and efficiency of these CDK inhibitors, a series of hybrid compounds called meriolins have been synthesized. METHODS: The potential antitumoral activity of meriolins was investigated in vitro on glioma cell lines (SW1088 and U87), native neural cells, and a human endothelial cell line (HUV-EC-C). The impact of intraperitoneal or intratumoral administrations of meriolin 15 was evaluated in vivo on 2 different nude mice-xenografted glioma models. RESULTS: Meriolins 3, 5, and 15 exhibited antiproliferative properties with nanomolar IC50 and induced cell-cycle arrest and CDK inhibition associated with apoptotic events in human glioma cell lines. These meriolins blocked the proliferation rate of HUV-EC-C through cell cycle arrest and apoptosis. In vivo, meriolin 15 provoked a robust reduction in tumor volume in spite of toxicity for highest doses, associated with inhibition of cell division, activation of caspase 3, reduction of CD133 cells, and modifications of the vascular architecture. CONCLUSION: Meriolins, and meriolin 15 in particular, exhibit antiproliferative and proapoptotic activities on both glioma and intratumoral endothelial cells, constituting key promising therapeutic lead compounds for the treatment of glioblastoma.

Down-regulation of GABA(A) receptor via promiscuity with the vasoactive peptide urotensin II receptor. Potential involvement in astrocyte plasticity.

GABA(A) receptor (GABA(A)R) expression level is inversely correlated with the proliferation rate of astrocytes after stroke or during malignancy of astrocytoma, leading to the hypothesis that GABA(A)R expression/activation may work as a cell proliferation repressor. A number of vasoactive peptides exhibit the potential to modulate astrocyte proliferation, and the question whether these mechanisms may imply alteration in GABA(A)R-mediated functions and/or plasma membrane densities is open. The peptide urotensin II (UII) activates a G protein-coupled receptor named UT, and mediates potent vasoconstriction or vasodilation in mammalian vasculature. We have previously demonstrated that UII activates a PLC/PIPs/Ca(2+) transduction pathway, via both G(q) and G(i/o) proteins and stimulates astrocyte proliferation in culture. It was also shown that UT/G(q)/IP(3) coupling is regulated by the GABA(A)R in rat cultured astrocytes. Here we report that UT and GABA(A)R are co-expressed in cerebellar glial cells from rat brain slices, in human native astrocytes and in glioma cell line, and that UII inhibited the GABAergic activity in rat cultured astrocytes. In CHO cell line co-expressing human UT and combinations of GABA(A)R subunits, UII markedly depressed the GABA current (ß(3)γ(2)>α(2)ß(3)γ(2)>α(2)ß(1)γ(2)). This effect, characterized by a fast short-term inhibition followed by drastic and irreversible run-down, is not relayed by G proteins. The run-down partially involves Ca(2+) and phosphorylation processes, requires dynamin, and results from GABA(A)R internalization. Thus, activation of the vasoactive G protein-coupled receptor UT triggers functional inhibition and endocytosis of GABA(A)R in CHO and human astrocytes, via its receptor C-terminus. This UII-induced disappearance of the repressor activity of GABA(A)R, may play a key role in the initiation of astrocyte proliferation.

Protective effect of the octadecaneuropeptide on hydrogen peroxide-induced oxidative stress and cell death in cultured rat astrocytes.

Oxidative stress, resulting from accumulation of reactive oxygen species (ROS), plays a critical role on astrocyte death associated with neurodegenerative diseases. Astroglial cells produce endozepines, a family of biologically active peptides that have been implicated in cell protection. Thus, the purpose of the present study was to investigate the potential protective effect of one of the endozepines, the octadecaneuropeptide ODN, on hydrogen peroxide (H(2) O(2) )-induced oxidative stress and cell death in rat astrocytes. Incubation of cultured astrocytes with graded concentrations of H(2) O(2) for 1 h provoked a dose-dependent reduction of the number of living cells as evaluated by lactate dehydrogenase assay. The cytotoxic effect of H(2) O(2) was associated with morphological modifications that were characteristic of apoptotic cell death. H(2) O(2) -treated cells exhibited high level of ROS associated with a reduction of both superoxide dismutases (SOD) and catalase activities. Pre-treatment of astrocytes with low concentrations of ODN dose-dependently prevented cell death induced by H(2) O(2) . This effect was accompanied by a marked attenuation of ROS accumulation, reduction of mitochondrial membrane potential and activation of caspase 3 activity. ODN stimulated SOD and catalase activities in a concentration-dependent manner, and blocked H(2) O(2) -evoked inhibition of SOD and catalase activities. Blockers of SOD and catalase suppressed the effect of ODN on cell survival. Taken together, these data demonstrate for the first time that ODN is a potent protective agent that prevents oxidative stress-induced apoptotic cell death.

The vasoactive peptides urotensin II and urotensin II-related peptide regulate astrocyte activity through common and distinct mechanisms: involvement in cell proliferation.

UII (urotensin II) and its paralogue URP (UII-related peptide) are two vasoactive neuropeptides whose respective central actions are currently unknown. In the present study, we have compared the mechanism of action of URP and UII on cultured astrocytes. Competition experiments performed with [125I]UII showed the presence of very-high- and high-affinity binding sites for UII, and a single high-affinity site for URP. Both UII and URP provoked a membrane depolarization accompanied by a decrease in input resistance, stimulated the release of endozepines, neuropeptides specifically produced by astroglial cells, and generated an increase in [Ca2+]c (cytosolic Ca2+ concentration). The UII/URP-induced [Ca2+]c elevation was PTX (pertussis toxin)-insensitive, and was blocked by the PLC (phospholipase C) inhibitor U73122 or the InsP3 channel blocker 2-APB (2-aminoethoxydiphenylborane). The addition of the Ca2+ chelator EGTA reduced the peak and abolished the plateau phase, whereas the T-type Ca2+ channel blocker mibefradil totally inhibited the Ca2+ response evoked by both peptides. However, URP and UII induced a mono- and bi-phasic dose-dependent increase in [Ca2+]c and provoked short- and long-lasting Ca2+ mobilization respectively. Similar mono- and bi-phasic dose-dependent increases in [3H]inositol incorporation into polyphosphoinositides in astrocytes was obtained, but the effect of UII was significantly reduced by PTX, although BRET (bioluminescence resonance energy transfer) experiments revealed that both UII and URP recruited Galphao-protein. Finally, UII, but not URP, exerted a dose-dependent mitogenic activity on astrocytes. Therefore we described that URP and UII exert not only similar, but also divergent actions on astrocyte activity, with UII exhibiting a broader range of activities at physiological peptide concentrations.

Beta-amyloid peptide stimulates endozepine release in cultured rat astrocytes through activation of N-formyl peptide receptors.

Astroglial cells synthesize and release endozepines, a family of neuropeptides derived from diazepam-binding inhibitor (DBI). The authors have recently shown that beta-amyloid peptide (Abeta) stimulates DBI gene expression and endozepine release. The purpose of this study was to determine the mechanism of action of Abeta in cultured rat astrocytes. Abeta(25-35) and the N-formyl peptide receptor (FPR) agonist N-formyl-Met-Leu-Phe (fMLF) increased the secretion of endozepines in a dose-dependent manner with EC(50) value of approximately 2 microM. The stimulatory effects of Abeta(25-35) and the FPR agonists fMLF and N-formyl-Met-Met-Met (fMMM) on endozepine release were abrogated by the FPR antagonist N-t-Boc-Phe-Leu-Phe-Leu-Phe. In contrast, Abeta(25-35) increased DBI mRNA expression through a FPR-independent mechanism. Abeta(25-35) induced a transient stimulation of cAMP formation and a sustained activation of polyphosphoinositide turnover. The stimulatory effect of Abeta(25-35) on endozepine release was blocked by the adenylyl cyclase inhibitor somatostatin, the protein kinase A (PKA) inhibitor H89, the phospholipase C inhibitor U73122, the protein kinase C (PKC) inhibitor chelerythrine and the ATP binding cassette transporter blocker glyburide. Taken together, these data demonstrate for the first time that Abeta(25-35) stimulates endozepine release from rat astrocytes through a FPR receptor positively coupled to PKA and PKC.

Effect of GABA A receptor activation on UT-coupled signaling pathways in rat cortical astrocytes.

Cultured rat cortical astrocytes express two types of urotensin II (UII) binding sites: a high affinity site corresponding to the UT (GPR14) receptor and a low affinity site that has not been fully characterized. Activation of the high affinity site in astroglial cells stimulates polyphosphoinositide (PIP) turnover and provokes an increase in intracellular calcium concentration. We have hypothesized that the existence of distinct affinity sites for UII in rat cortical astrocytes could be accounted for by a possible cross-talk between UT and the ligand-gated ion channel GABA(A) receptor (GABA A R). Exposure of cultured astrocytes to UII provoked a bell-shaped increase in cAMP production, with an EC50 stimulating value of 0.83+/-0.04 pM, that was totally blocked in the presence of the adenylyl cyclase inhibitor SQ 22,536. In contrast, UII was found to inhibit forskolin-induced cAMP formation. In the presence of the specific PKA inhibitor H89, UII provoked a sustained stimulation of cAMP formation. Inhibition of PKA by H89 strongly reduced the stimulatory effect of UII on PIP metabolism. GABA and the GABA A R agonist isoguvacine provoked a marked inhibition of UII-induced cAMP synthesis and a significant reduction of UII-evoked PIP turnover. These data suggest that functional interaction between UT and GABA(A)R negatively regulates coupling of UT to the classical PLC/IP(3) signaling cascade as well as to the adenylyl cyclase/PKA pathway.

[Orn5]URP acts as a pure antagonist of urotensinergic receptors in rat cortical astrocytes.

Cultured rat astrocytes, which express functional urotensin II (UII)/UII-related peptide (URP) receptors (UT), represent a very suitable model to investigate the pharmacological profile of UII and URP analogs towards native UT. We have recently designed three URP analogs [D-Trp4]URP, [Orn5]URP and [D-Tyr6]URP, that act as UT antagonists in the rat aortic ring bioassay. However, it has been previously reported that UII/URP analogs capable of inhibiting the contractile activity of UII possess agonistic activity on UT-transfected cells. In the present study, we have compared the ability of URP analogs to compete for [125 I]URP binding and to modulate cytosolic calcium concentration ([Ca2+]c) in cultured rat astrocytes. All three analogs displaced radioligand binding: [D-Trp4]URP and [D-Tyr6]URP interacted with high- and low-affinity sites whereas [Orn5]URP only bound high-affinity sites. [D-Trp4]URP and [D-Tyr6]URP both induced a robust increase in [Ca2+]c in astrocytes while [Orn5]URP was totally devoid of activity. [Orn5]URP provoked a concentration-dependent inhibition of URP- and UII-evoked [Ca2+]c increase and a rightward shift of the URP and UII dose-response curves. The present data indicate that [D-Trp4]URP and [D-Tyr6]URP, which act as UII antagonists in the rat aortic ring assay, behave as agonists in the [Ca2+]c mobilization assay in cultured astrocytes, whereas [Orn5]URP is a pure selective antagonist in both rat aortic ring contraction and astrocyte [Ca2+]c mobilization assays.