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Background and Aims: To better understand nonalcoholic steatohepatitis (NASH) disease progression and to evaluate drug targets and compound activity, we undertook the development of an in vitro 3D model to mimic liver architecture and the NASH environment. Methods: We have developed an in vitro preclinical 3D NASH model by coculturing primary human hepatocytes, human stellate cells, liver endothelial cells and Kupffer cells embedded in a hydrogel of rat collagen on a 96-well plate. A NASH-like environment was induced by addition of medium containing free fatty acids and tumor necrosis factor-α. This model was then characterized by biochemical, imaging and transcriptomics analyses. Results: We succeeded in defining suitable culture conditions to maintain the 3D coculture for up to 10 days in vitro , with the lowest level of steatosis and reproducible low level of inflammation and fibrosis. NASH disease was induced with a custom medium mimicking NASH features. The cell model exhibited the key NASH disease phenotypes of hepatocyte injury, steatosis, inflammation, and fibrosis. Hepatocyte injury was highlighted by a decrease of CYP3A4 expression and activity, without loss of viability up to day 10. Moreover, the model was able to stimulate a stable inflammatory and early fibrotic environment, with expression and secretion of several cytokines. A global gene expression analysis confirmed the NASH induction. Conclusions: This is a new in vitro model of NASH disease consisting of four human primary cell-types that exhibits most features of the disease. The 10-day cell viability and cost effectiveness of the model make it suitable for medium throughput drug screening and provide attractive avenues to better understand disease physiology and to identify and characterize new drug targets., Competing Interests: All of the authors except TS were employees of Sanofi during the course of the study, and all are or have been shareholders of Sanofi., (© 2020 Authors.)

This article describes the chemical synthesis, ADME and pharmacological properties and early safety pharmacology evaluation of a series of novel Nurr1/NOT agonist. It is meant as a support to an article recently published in Bioorganic and Medicinal chemistry Letters and entitled "Development of a novel NURR1/NOT agonist from hit to lead and candidate for the potential treatment of Parkinson's disease" [1] and presenting the discovery, scope and potential of these new ligands of these nuclear receptors., (© 2019 The Authors.)

In the course of a programme aimed at identifying Nurr1/NOT agonists for potential treatment of Parkinson's disease, a few hits from high throughput screening were identified and characterized. A combined optimization pointed to a very narrow and stringent structure activity relationship. A comprehensive program of optimization led to a potent and safe candidate drug displaying neuroprotective and anti-inflammatory activity in several in vitro and in vivo models., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The use of colloidal silica nanoparticles and sub-microparticles (SiPs) have been considered a very interesting strategy for drug delivery applications. In the present study, we have focused our attention on the suitability of these nanomaterials as potential carriers for dermal drug delivery, thus studying their toxicological profile in vitro, cellular uptake and intracellular localization in both human keratinocytes (K17) and human dermal fibroblasts (HDF) as a function of their particle size (SiPs of 20, 70, 200 and 500 nm). Full characterization of these aspects enabled us to observe a strong cell-type dependency in terms of cytotoxicity and cell internalization, whereas particle size was only relevant for ultra-small SiPs (20 nm), being the most toxic SiPs. For 70, 200 and 500 nm SiPs, the differences in uptake and intracellular trafficking determined the different toxicological profiles in K17 and HDF. In addition, these characteristics can further define different drug delivery strategies. Hence, phagocytosis has been identified as the main internalization mechanism for K17, and caveolae-mediated endocytosis for HDF. This relevant information led us to conclude that fibroblasts would be optimal targets for delivering delicate therapeutic molecules such as proteins or genetic material using SiPs while maintaining a low toxicity profile, whereas keratinocytes could enable accelerated drug release therapies based on SiPs., (Copyright © 2017 John Wiley & Sons, Ltd.)

Normalization of altered glutamate neurotransmission through activation of the mGluR2 has emerged as a new approach to treat schizophrenia. These studies describe a potent brain penetrant mGluR2 positive allosteric modulator (PAM), SAR218645. The compound behaves as a selective PAM of mGluR2 in recombinant and native receptor expression systems, increasing the affinity of glutamate at mGluR2 as inferred by competition and GTPγ 35 S binding assays. SAR218645 augmented the mGluR2-mediated response to glutamate in a rat recombinant mGluR2 forced-coupled Ca 2+ mobilization assay. SAR218645 potentiated mGluR2 agonist-induced contralateral turning. When SAR218645 was tested in models of the positive symptoms of schizophrenia, it reduced head twitch behavior induced by DOI, but it failed to inhibit conditioned avoidance and hyperactivity using pharmacological and transgenic models. Results from experiments in models of the cognitive symptoms associated with schizophrenia showed that SAR218645 improved MK-801-induced episodic memory deficits in rats and attenuated working memory impairment in NMDA Nr1 neo-/- mice. The drug reversed disrupted latent inhibition and auditory-evoked potential in mice and rats, respectively, two endophenotypes of schizophrenia. This profile positions SAR218645 as a promising candidate for the treatment of cognitive symptoms of patients with schizophrenia, in particular those with abnormal attention and sensory gating abilities., Competing Interests: Organizations from whom the authors have received compensation for professional services: Guy Griebel, employee of Sanofi; Philippe Pichat, employee of Sanofi; Vanessa Naimoli, former employee of Sanofi; Lisa Potestio, former employee of Sanofi; Robert Featherstone, former employee of Sanofi; Sukhveen Sahni, former employee of Sanofi; Henry Defex, former employee of Sanofi; Christophe Desvignes, employee of Sanofi; Franck Slowinski, employee of Sanofi; Xavier Vigé, employee of Sanofi; Olivier E. Bergis, employee of Sanofi; Rosy Sher, former employee of Sanofi; Raymond Kosley, former employee of Sanofi; Sathapana Kongsamut, former employee of Sanofi; Mark D. Black, former employee of Sanofi; Geoffrey B. Varty, former employee of Sanofi. Drs Kosley and Sher are inventors on a patent covering the main product described in this report.

E-prostanoid receptor subtype 2 (EP2) agonists are currently under clinical development as hypotensive agents for the treatment of ocular hypertension. However, the effects of EP2 receptor agonists on trabecular meshwork (TM) alterations leading to primary open-angle glaucoma (POAG) are still unknown. Here, we evaluated whether EP2 receptor activation exhibits protective functions on TM cell death induced by endoplasmic reticulum (ER) stress. We show that the EP2 receptor agonist butaprost protects TM cell death mediated by the ER stress inducer tunicamycin through a cyclic AMP (cAMP)-dependent mechanism, but independent of the classical cAMP sensors, protein kinase A and exchange proteins activated by cAMP. The ER stress-induced intrinsic apoptosis inhibited by the EP2 receptor agonist was correlated with a decreased accumulation of the cellular stress sensor p53. In addition, p53 down-regulation was associated with inhibition of its transcriptional activity, which led to decreased expression of the pro-apoptotic p53-upregulated modulator of apoptosis (PUMA). The stabilization of p53 by nutlin-3a abolished butaprost-mediated cell death protection. In conclusion, we showed that EP2 receptor activation protects against ER stress-dependent mitochondrial apoptosis through down-regulation of p53. The specific inhibition of this pathway could reduce TM alterations observed in POAG patients., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Purpose: Prostaglandin F2α analogues are the first-line medication for the treatment of ocular hypertension (OHT), and prostanoid EP2 receptor agonists are under clinical development for this indication. The goal of this study was to investigate the effects of F prostanoid (FP) and EP2 receptor activation on the myofibroblast transition of primary trabecular meshwork (TM) cells, which could be a causal mechanism of TM dysfunction in glaucoma., Methods: Human primary TM cells were treated with either latanoprost or butaprost and TGF-β2. Trabecular meshwork contraction was measured in a three-dimensional (3D) TM cell-populated collagen gel (CPCG) model. Expression of α-smooth muscle actin (α-SMA) and phosphorylation of myosin light chain (MLC) were determined by Western blot. Assembly of actin stress fibers and collagen deposition were evaluated by immunocytochemistry. Involvement of p38, extracellular signal-regulated kinase (ERK), and Rho-associated kinase (ROCK) pathways as well as matrix metalloproteinase activation was tested with specific inhibitors., Results: In one source of validated adult TM cells, latanoprost induced cell contraction as observed by CPCG surface reduction and increased actin polymerization, α-SMA expression, and MLC phosphorylation, whereas butaprost inhibited TGF-β2-induced CPCG contraction, actin polymerization, and MLC phosphorylation. Both agonists inhibited TGF-β2-dependent collagen deposition. The latanoprost effects were mediated by p38 pathway., Conclusions: Latanoprost decreased TM collagen accumulation but promoted a contractile phenotype in a source of adult TM cells that could modulate the conventional outflow pathway. In contrast, butaprost attenuated both TM contraction and collagen deposition induced by TGF-β2, thereby inhibiting myofibroblast transition of TM cells. These results open new perspectives for the management of OHT.

On native human, rat and mouse glycine transporter-1(GlyT1), SSR130800 behaves as a selective inhibitor with IC50 values of 1.9, 5.3 and 6.8 nM, respectively. It reversibly blocked glycine uptake in mouse brain cortical homogenates, increased extracellular levels of glycine in the rat prefrontal cortex, and potentiated NMDA-mediated excitatory postsynaptic currents in rat hippocampal slices. SSR103800 (30 mg/kg, p.o.) decreased MK-801- and PCP-induced locomotor hyperactivity in rodents. SSR103800 (1 and 10 mg/kg, p.o.) attenuated social recognition deficit in adult rats induced by neonatal injections of PCP (10 mg/kg, s.c., on post-natal day 7, 9 and 11). SSR103800 (3 mg/kg, p.o.) counteracted the deficit in short-term visual episodic-like memory induced by a low challenge dose of PCP (1 mg/kg, i.p.), in PCP-sensitized rats (10 mg/kg, i.p.). SSR103800 (30 mg/kg, i.p.) increased the prepulse inhibition of the startle reflex in DBA/1J mice. SSR103800 decreased defensive- and despair-related behaviors in the tonic immobility test in gerbils (10 and 30 mg/kg, p.o.) and in the forced-swimming procedure in rats (1 and 3 mg/kg, p.o.), respectively. These findings suggest that SSR103800 may have a therapeutic potential in the management of the core symptoms of schizophrenia and comorbid depression states.

In this paper, we report on the pharmacological and functional profile of SSR180711 (1,4-Diazabicyclo[3.2.2]nonane-4-carboxylic acid, 4-bromophenyl ester), a new selective alpha7 acetylcholine nicotinic receptor (n-AChRs) partial agonist. SSR180711 displays high affinity for rat and human alpha7 n-AChRs (K(i) of 22+/-4 and 14+/-1 nM, respectively). Ex vivo (3)[H]alpha-bungarotoxin binding experiments demonstrate that SSR180711 rapidly penetrates into the brain (ID(50)=8 mg/kg p.o.). In functional studies performed with human alpha7 n-AChRs expressed in Xenopus oocytes or GH4C1 cells, the compound shows partial agonist effects (intrinsic activity=51 and 36%, EC(50)=4.4 and 0.9 microM, respectively). In rat cultured hippocampal neurons, SSR180711 induced large GABA-mediated inhibitory postsynaptic currents and small alpha-bungarotoxin sensitive currents through the activation of presynaptic and somato-dendritic alpha7 n-AChRs, respectively. In mouse hippocampal slices, the compound increased the amplitude of both glutamatergic (EPSCs) and GABAergic (IPSCs) postsynaptic currents evoked in CA1 pyramidal cells. In rat and mouse hippocampal slices, a concentration of 0.3 muM of SSR180711 increased long-term potentiation (LTP) in the CA1 field. Null mutation of the alpha7 n-AChR gene totally abolished SSR180711-induced modulation of EPSCs, IPSCs and LTP in mice. Intravenous administration of SSR180711 strongly increased the firing rate of single ventral pallidum neurons, extracellularly recorded in anesthetized rats. In microdialysis experiments, administration of the compound (3-10 mg/kg i.p.) dose-dependently increased extracellular acetylcholine (ACh) levels in the hippocampus and prefrontal cortex of freely moving rats. Together, these results demonstrate that SSR180711 is a selective and partial agonist at human, rat and mouse alpha7 n-AChRs, increasing glutamatergic neurotransmission, ACh release and LTP in the hippocampus.

Noncompetitive N-methyl-D-aspartate (NMDA) blockers induce schizophrenic-like symptoms in humans, presumably by impairing glutamatergic transmission. Therefore, a compound potentiating this neurotransmission, by increasing extracellular levels of glycine (a requisite co-agonist of glutamate), could possess antipsychotic activity. Blocking the glycine transporter-1 (GlyT1) should, by increasing extracellular glycine levels, potentiate glutamatergic neurotransmission. SSR504734, a selective and reversible inhibitor of human, rat, and mouse GlyT1 (IC50=18, 15, and 38 nM, respectively), blocked reversibly the ex vivo uptake of glycine (mouse cortical homogenates: ID50: 5 mg/kg i.p.), rapidly and for a long duration. In vivo, it increased (minimal efficacious dose (MED): 3 mg/kg i.p.) extracellular levels of glycine in the rat prefrontal cortex (PFC). This resulted in an enhanced glutamatergic neurotransmission, as SSR504734 potentiated NMDA-mediated excitatory postsynaptic currents (EPSCs) in rat hippocampal slices (minimal efficacious concentration (MEC): 0.5 microM) and intrastriatal glycine-induced rotations in mice (MED: 1 mg/kg i.p.). It normalized activity in rat models of hippocampal and PFC hypofunctioning (through activation of presynaptic CB1 receptors): it reversed the decrease in electrically evoked [3H]acetylcholine release in hippocampal slices (MEC: 10 nM) and the reduction of PFC neurons firing (MED: 0.3 mg/kg i.v.). SSR504734 prevented ketamine-induced metabolic activation in mice limbic areas and reversed MK-801-induced hyperactivity and increase in EEG spectral energy in mice and rats, respectively (MED: 10-30 mg/kg i.p.). In schizophrenia models, it normalized a spontaneous prepulse inhibition deficit in DBA/2 mice (MED: 15 mg/kg i.p.), and reversed hypersensitivity to locomotor effects of d-amphetamine and selective attention deficits (MED: 1-3 mg/kg i.p.) in adult rats treated neonatally with phencyclidine. Finally, it increased extracellular dopamine in rat PFC (MED: 10 mg/kg i.p.). The compound showed additional activity in depression/anxiety models, such as the chronic mild stress in mice (10 mg/kg i.p.), ultrasonic distress calls in rat pups separated from their mother (MED: 1 mg/kg s.c.), and the increased latency of paradoxical sleep in rats (MED: 30 mg/kg i.p.). In conclusion, SSR504734 is a potent and selective GlyT1 inhibitor, exhibiting activity in schizophrenia, anxiety and depression models. By targeting one of the primary causes of schizophrenia (hypoglutamatergy), it is expected to be efficacious not only against positive but also negative symptoms, cognitive deficits, and comorbid depression/anxiety states.

The biochemical and pharmacological properties of a novel antagonist of the tachykinin neurokinin 1 (NK1) receptor, SSR240600 [(R)-2-(1-[2-[4-[2-[3,5-bis(trifluoromethyl)phenyl]acetyl]-2-(3,4-dichlorophenyl)-2-morpholinyl]ethyl]-4-piperidinyl)-2-methylpropanamide], were evaluated. SSR240600 inhibited the binding of radioactive substance P to tachykinin NK1 receptors in human lymphoblastic IM9 cells (K(i) = 0.0061 nM), human astrocytoma U373MG cells (K(i) = 0.10 nM), and human brain cortex (IC50 = 0.017 nM). It also showed subnanomolar affinity for guinea pig NK1 receptors but was less potent on rat and gerbil NK1 receptors. SSR240600 inhibited [Sar(9),Met(O2)(11)]substance P-induced inositol monophosphate formation in human astrocytoma U373MG cells with an IC50 value of 0.66 nM (agonist concentration of 100 nM). It also antagonized substance P-induced contractions of isolated human small bronchi with a pIC50 value of 8.6 (agonist concentration of 100 nM). The compound was >100- to 1000-fold more selective for tachykinin NK1 receptors versus tachykinin NK2 or NK3 receptors as evaluated in binding and in vitro functional assays. In vivo antagonistic activity of SSR240600 was demonstrated on tachykinin NK1 receptor-mediated hypotension in dogs (3 and 10 microg/kg i.v.), microvascular leakage (1 and 3 mg/kg i.p.), and bronchoconstriction (50 and 100 microg/kg i.v.) in guinea pigs. It also prevented citric acid-induced cough in guinea pigs (1-10 mg/kg i.p.), an animal model in which central endogenous tachykinins are suspected to play a major role. In conclusion, SSR240600 is a new, potent, and centrally active antagonist of the tachykinin NK1 receptor, able to antagonize various NK1 receptor-mediated pharmacological effects in the periphery and in the central nervous system.

Among the chemokine family, fractalkine shows unusual properties: it exists as a membrane-bound and soluble protein, and both fractalkine and its receptor CX(3)CR1 are expressed predominantly in the central nervous system. In rat cell culture models, the chemokine fractalkine was expressed in neurons and microglia, but not in astrocytes and its receptor exclusively localized to microglial cells, where its expression was downregulated by treatment with the bacterial endotoxin (LPS). In microglial cultures, LPS (10 ng/ml) induced a marked increase in the release of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). The effects of LPS on TNF-alpha secretion were partially blocked (30%) by fractalkine and the effects of fractalkine were reversed by a polyclonal anti-fractalkine antibody. When microglial-associated fractalkine was neutralized by anti-fractalkine antibody, the LPS response was increased by 80%, suggesting tonic activation of microglial fractalkine receptors by endogenous fractalkine. The effects of the antibody were antagonized by the addition of fractalkine. LPS-activated microglia were neurotoxic when added to neuronal hippocampal culture, producing 20% neuronal death, as measured by NeuN-positive cell counting. An anti-fractalkine antibody produced neurotoxic effects of similar magnitude in this co-culture system and also markedly potentiated the neurotoxic effects of LPS-activated microglia (40% neuronal death). These results suggest that endogenous fractalkine might act tonically as an anti-inflammatory chemokine in cerebral tissue through its ability to control and suppress certain aspects of microglial activation. These data may have relevance to degenerative conditions such as multiple sclerosis, in which cerebral inflammatory processes may be activated., (Copyright 2000 Wiley-Liss, Inc.)

In situ hybridization studies have revealed high levels of protease (thrombin)-activated receptor-1 messenger RNA in the mesencephalon of rats, suggesting that dopaminergic neurons are a target for thrombin's actions. We have evaluated the effect of thrombin receptor activation, either by thrombin or by thrombin receptor agonist peptide, a 14 amino acid agonist of protease-activated receptor-1, on tyrosine hydroxylase-positive neurons. Pure cultures of rat mesencephalic neurons or co-cultures of mesencephalic neurons and glial cells were treated with either thrombin or thrombin receptor agonist peptide the day after plating. Tyrosine hydroxylase-positive cell counting, [3H]dopamine uptake and morphometric analysis were performed on day 5. Thrombin and thrombin receptor agonist peptide influenced neurite elongation, branching and the number of primary, secondary and tertiary neurites of tyrosine hydroxylase-positive neurons. In pure cultures, the most significant effects of thrombin and thrombin receptor agonist peptide were to delay branching and to increase the centrifugal growth of neurites without affecting the total neuritic length. Thrombin (up to 10 nM) and thrombin receptor agonist peptide did not affect the number of tyrosine hydroxylase-positive neurons or [3H]dopamine uptake. Neurotrophin-4 also influenced the morphology of tyrosine hydroxylase-positive neurons. The increase of neuritic length initiated by this neurotrophin is complementary to the radial elongation induced by protease-activated receptor-1 activation. When neurons were cultured in the presence of glial cells, the effects of thrombin and thrombin receptor agonist peptide on most of these parameters were larger than those observed with pure cultures. Thus, thrombin is able to initiate a complex remodelling of the architecture of tyrosine hydroxylase-positive neurons through the activation of protease-activated receptor-1. These results provide further support for the involvement of protease-activated receptor-1 activation in the development and differentiation of the central nervous system.

There is increasing evidence that immunophilins play a role in neural development and differentiation. We have studied the neurotrophic effects of FK-506, rapamycin and cyclosporin A (CsA) on dorsal root ganglia (DRG) taken from different segmental levels (cervical, thoracic and lumbar/sacral), and on rat embryonic septal cholinergic neurons in culture. At a low concentration (1 nM), FK-506 significantly increased (+83%) the number of neurites of thoracic DRG explants. At a higher concentration (100 nM), it also enhanced the neuritogenesis of thoracic (+100%) and lumbar/sacral (+57%) DRG, but not cervical DRG explants. Rapamycin displayed a converse effect, reducing the development of DRG explants from cervical and thoracic segments (-78% at 1 nM in thoracic DRG). CsA (from 1 to 100 nM) was without effect on DRG neuritogenesis. In contrast to nerve growth factor (NGF), which increased neurite length (+116% at 3 ng/ml), neither FK-506 nor rapamycin affected this parameter.

The proteolytic action of thrombin on its receptor (protease-activated receptor-1 or PAR-1) results in a conformational change in which the new N-terminal sequence auto-activates the receptor. Peptide analogs of this N-terminal sequence (TRAPs) are able to mimic the effect of thrombin and an extensive search has led to the definition of the structural requirement for the agonist and antagonist activity on thrombin receptors in several peripheral systems. Thrombin plays an important role in central and peripheral nervous system development and PAR-1 is present in neurons and astrocytes. We have now characterized thrombin receptors pharmacologically in cultured rat astrocytes by using [3H]thymidine incorporation and reversal of stellation induced by Bt2cAMP as end-points. Thrombin increased [3H]thymidine incorporation into DNA with an EC50 of 1 nM and induced a complete reversion of cell stellation. The effects of thrombin on [3H]thymidine incorporation were mimicked by TRAP-14 (EC50 = 3 microM) and a peptide containing non-natural amino acids Ala-Phe(p-F)-Arg-Cha-HArg-Tyr-NH2 (A6Y; EC50 = 0.8 microM). Similarly, these two peptides reversed Bt2cAMP-induced stellation. The effect of thrombin, TRAP-14 and A6Y on [3H]thymidine incorporation into DNA was significantly prevented by L9R, a 9-amino-acid peptide (Leu-Val-Arg-D-Cys-Gly-Lys-His-Ser-Arg; IC50 = 180 microM against thrombin and TRAP-14 and 800 microM against A6Y) previously described as an antagonist in human platelet aggregation. L9R antagonized also thrombin effects on astrocyte morphology. These results demonstrate that rat astrocytes express PAR-1 receptors which are pharmacologically similar to those previously characterized in human platelets.

In astrocytes, thrombin and thrombin receptor-activating peptide (TRAP-14), a 14-amino-acid agonist of the proteolytic activating receptor for thrombin (PART), significantly increased cell division as assessed by [3H]-thymidine incorporation into DNA (EC50 = 1 nM and +650% at 100 nM for thrombin; EC50 = 3 microM and +600% at 100 microM for TRAP-14) and nerve growth factor (NGF) secretion (approximately twofold at 100 nM thrombin or 100 microM TRAP-14). The [3H] thymidine incorporation was prevented by protein kinase C inhibitors (staurosporine and H7) or by down-regulation of this enzyme by chronic exposure of astrocytes to phorbol 12-myristate 13-acetate (PMA). Thrombin-induced NGF secretion was completely inhibited by protein kinase C inhibitors. Treatment with PMA stimulated NGF secretion 19-fold, and this effect was not further enhanced by thrombin. These data suggest an absolute requirement of protein kinase C activity for thrombin-induced NGF secretion and cell division. Pretreatment of astrocytes with pertussis toxin (PTX) reduced thrombin- and TRAP-14-induced DNA synthesis. PART activation caused a decrease in forskolin-stimulated cyclic AMP accumulation. PTX treatment prevented the inhibitory effect of PART activation on cyclic AMP accumulation, suggesting that a PTX-sensitive G protein, such as Gi or G(o), is involved in thrombin-induced cell division. In contrast, thrombin-induced NGF secretion was not inhibited by PTX. Finally, the protein tyrosine kinase inhibitor herbimycin A partially but significantly prevented thrombin- and TRAP-14-induced cell division but was without effect on NGF secretion. Taken together, these results demonstrate that, in astrocytes, PART(s)-triggered cell division or NGF secretion is mediated by distinct transduction mechanisms.

We have compared the effects of thrombin and of the 14-amino acid peptide agonist (TRAP-14) of the thrombin protease activated receptor (PAR) on cholinergic neurons in pure cultures of rat septal neurons and in co-cultures of septal neurons and glial cells. In pure septal cultures, low concentrations of thrombin (up to 10 nM) did not affect choline acetyltransferase (ChAT) activity, a marker of cholinergic neurons, or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction, an index of cell viability. However, 100 nM thrombin decreased ChAT activity and MTT reduction by 44 and 17%, respectively. In co-cultures, a low concentration of thrombin (1 nM) increased ChAT activity (+75%), whereas a high concentration (100 nM) decreased it (-83%). At this high concentration, thrombin was neurotoxic, as indicated by a large decrease in MTT reduction (-80%). Thrombin effects on ChAT activity were mimicked by TRAP-14 both in pure septal cultures (no effect at 0.1 microM and -63% at 100 microM) and in co-cultures (+25% at 0.1 microM and -28% at 100 microM). In contrast, this peptide did not affect MTT reduction. These dual effects of thrombin and TRAP-14 on ChAT activity in co-cultures, were also observed on pure cultures of septal cells supplied with NGF. The activation and inhibition by TRAP-14 of the expression of ChAT activity in septal neuron/glial cell cultures were inhibited by a 9-amino acid peptide antagonist of thrombin PAR. Thus, the effects of thrombin on cholinergic neurons seem to be mainly mediated by thrombin PAR and glial cells seem to play a major role in these thrombin actions.

The neuroprotective effects of various doses of N omega-nitro-L-arginine have been correlated with the degree of N omega-nitro-L-arginine-induced inhibition of cortical nitric oxide synthase activity measured ex vivo. Following focal cerebral ischemia induced by permanent occlusion of middle cerebral artery in the mouse, repeated administration of 1 mg/kg i.p. of N omega-nitro-L-arginine (beginning 5 min after surgery) reproducibly decreased by 66-76% the infarct volume measured at 6 days post-occlusion. This dose of N omega-nitro-L-arginine decreased cortical nitric oxide (NO) synthase activity by 70-73%. The neuroprotective efficacy of N omega-nitro-L-arginine increased dose-dependently over the range of doses of 0.1-1 mg/kg. Within this dose range of N omega-nitro-L-arginine, there was a good parallelism between the extent of inhibition of cortical NO synthase activity measured ex vivo and the degree of neuroprotection. However, higher doses of N omega-nitro-L-arginine (3 and 10 mg/kg i.p.), which inhibited NO synthase activity more effectively (up to 94%) failed to significantly reduce the infarct size. Repeated administrations of increasing doses of L-arginine (up to 30 mg/kg i.p.) with a low dose of N omega-nitro-L-arginine (1 mg/kg i.p.) caused a dose-dependent reduction in the neuroprotective efficacy of N omega-nitro-L-arginine while the extent of NO synthase inhibition measured ex vivo did not decrease significantly.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of NG-nitro-L-arginine on L-glutamate-induced neurotoxicity have been evaluated on primary cultures of neonatal rat cortical neurons. Treatment of cultures with increasing concentrations of L-glutamate during 5 min produced a delayed neuronal death, as measured by lactate dehydrogenase release in the medium 24 h later. Maximal toxicity was obtained with 500 microM of L-glutamate. Substantial nitric oxide synthase activity was detected in these cortical cultures. Nitric oxide synthase activity and cellular L-glutamate-induced cyclic guanosine 3',5'-monophosphate accumulation were totally inhibited by 100 microM NG-nitro-L-arginine. Addition of NG-nitro-L-arginine (100 microM) to the medium either 5 min prior to and during L-glutamate exposure (500 microM, 5 min) or for 24 h after L-glutamate exposure decreased the amino acid-induced neurotoxicity by 23% (not significant) and 43%, respectively. When added 5 min before L-glutamate and just after L-glutamate removal and kept in contact with neurons for the following 24 h, NG-nitro-L-arginine (100 microM) antagonized by 74% the L-glutamate-induced neurotoxicity. This effect was not reversed by a co-application of L-arginine (1 mM). The neuroprotective effect of NG-nitro-L-arginine was concentration-dependent, a half-maximal inhibition of L-glutamate-induced neurotoxicity being observed with the addition (before and after L-glutamate) of 4 microM of the drug. These results suggest that the neuroprotective effect of NG-nitro-L-arginine previously observed in vivo is exerted at the neuronal level.(ABSTRACT TRUNCATED AT 250 WORDS)

Primary cultures of neonatal rat cortical neurons and astrocytes synthesize and secrete nerve growth factor (NGF). Co-culturing neurons with astrocytes decreased NGF secretion in the co-cultures. The inhibition of co-culture NGF secretion was partially reversible upon selectively decreasing the number of neurons by glutamate treatment. Interleukin-1 beta (IL-1) stimulated NGF secretion from astrocytes, and the magnitude of this secretion was decreased in the co-cultures. Thus, co-culture with neurons decreases astroglial cell secretion of NGF and down-regulates astroglial responsiveness to IL-1.

Neonatal rat cortical astrocytes in primary culture synthesize and secrete nerve growth factor (NGF). Interleukin-1 beta(IL-1) and basic fibroblast growth factor (bFGF) treatment of astrocytes increased NGF mRNA content by about 2-fold. The effect of these two factors was specific, because other growth factors, such as tumor necrosis factor-alpha, insulin-like growth factor-1, and epidermal growth factor, failed to change NGF mRNA content. The concentrations of IL-1 and bFGF causing half-maximal stimulation were 1 unit/ml and 1 ng/ml, respectively. The increase in NGF mRNA elicited by IL-1 and bFGF was maximal at 3 hr of incubation. In the presence of IL-1 this increase persisted for 36 hr, whereas in the presence of bFGF the initial increase in NGF mRNA was followed by a decrease to 50% of control levels after 24 hr of incubation. Readdition of bFGF after 24 hr of treatment gave a similar increase in NGF mRNA content, suggesting that the decrease at 24 hr was not due to receptor desensitization. The effect of IL-1 was reversible, because removal of IL-1 after 3 hr of incubation resulted in a decrease of NGF mRNA content to control levels by 6 hr, whereas a readdition of IL-1 at this time led to a 2-3-fold increase in NGF mRNA content after an additional 3 hr of treatment. This second increase in NGF mRNA was also maintained for several hours. The combined treatment of astrocytes with maximally effective doses of IL-1 and bFGF produced an additive increase in NGF mRNA content, suggesting that different mechanisms are operative. Treatment of astrocytes with cycloheximide increased (about 6-fold) NGF mRNA content, and this content failed to increase further with IL-1 or bFGF treatment. Experiments using actinomycin D indicated that IL-1 increased the stability of the NGF mRNA. bFGF treatment failed to change this parameter. Thus, IL-1 increases NGF mRNA content in astrocytes, at least in part, by stabilizing mRNA, whereas bFGF does not affect mRNA stability but may act at the level of NGF gene transcription.

Primary cultures of neonatal rat cortical astrocytes contain low cellular levels (about 2 pg/mg of protein) of nerve growth factor (NGF), but secrete significant amounts of NGF into the culture medium (about 540 pg of NGF/mg of cell protein/38-h incubation). Incubation of astrocytes with interleukin-1 (IL-1) increased the cellular content of NGF and the amount secreted by about threefold. In comparison, cerebellar astrocytes secreted significant amounts of NGF, and the secretion was also stimulated by IL-1. The stimulatory action of IL-1 on astrocytes prepared from cortex was dose- and time-dependent. Concentrations of IL-1 causing half-maximal and maximal stimulation of NGF secretion were 1 and 10 U/ml, respectively). Maximal NGF secretion induced by IL-1 (10 U/ml) was seen following 38 h of incubation. The basal secretion of NGF was reduced by about 50% under Ca2(+)-free conditions; however, the percent stimulation of NGF secretion by IL-1 was the same in the absence or presence of Ca2+. The stimulatory action of IL-1 was specific, because other glial growth factors and cytokines were almost ineffective in stimulating NGF secretion from cortical astroglial cells. IL-1 treatment also increased cellular NGF mRNA content twofold. The results indicate that IL-1 specifically triggers a cascade of events, independent of cell growth, which regulate NGF mRNA content and NGF secretion by astrocytes.