Nitric oxide-cyclic GMP signaling pathway limits inflammatory degeneration of midbrain dopaminergic neurons: cell type-specific regulation of heme oxygenase-1 expression.

Excessive production of nitric oxide (NO) by microglia is at least in part responsible for the pathogenesis of various neurodegenerative disorders including Parkinson disease, but at the same time NO may also play a distinct role as a signaling molecule such as an activator of soluble guanylyl cyclase. Here we investigated potential roles of the NO-soluble guanylyl cyclase-cyclic GMP signaling pathway in the regulation of dopaminergic neurodegeneration. Activation of microglia by interferon-gamma (IFN-gamma) followed by lipopolysaccharide (LPS) caused dopaminergic cell death in rat midbrain slice cultures, which was dependent on NO production. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a soluble guanylyl cyclase inhibitor, as well as KT5823, an inhibitor of cyclic GMP-dependent protein kinase, exacerbated dopaminergic cell death induced by IFN-gamma/LPS. Conversely, 8-bromo-cyclic GMP attenuated IFN-gamma/LPS cytotoxicity on dopaminergic neurons. Notably, although heme oxygenase-1 (HO-1) was expressed prominently in cells other than dopaminergic neurons in control cultures, robust expression of HO-1 was induced in surviving dopaminergic neurons challenged with IFN-gamma/LPS. ODQ and KT5823 decreased, whereas 8-bromo-cyclic GMP increased, the number of dopaminergic neurons expressing HO-1 after IFN-gamma/LPS challenge, without parallel changes in HO-1 expression in other cell populations. An NO donor 3-(4-morpholinyl)sydnonimine hydrochloride also induced HO-1 expression in dopaminergic neurons, which was abolished by ODQ and augmented by 8-bromo-cyclic GMP. Moreover, IFN-gamma/LPS-induced dopaminergic cell death was augmented by zinc protoporphyrin IX, an HO-1 inhibitor. The NO donor cytotoxicity on dopaminergic neurons was also augmented by ODQ and zinc protoporphyrin IX. These results indicate that the NO-cyclic GMP signaling pathway promotes the induction of HO-1 specifically in dopaminergic neurons, which acts as an endogenous protective system to limit inflammatory degeneration of this cell population.

Glucocorticoid enhances the response of type II cells from newborn rats to surfactant secretagogues.

There is a developmental increase in agonist-induced surfactant secretion in type II cells. The response to the P2Y(2) agonist UTP is negligible in early newborn cells but increases with age. The response to terbutaline, N-ethylcarboxyamidoadenosine (NECA), and ATP also increases with age. As glucocorticoids are known to accelerate several aspects of lung maturation we examined the effect of dexamethasone (Dex) on the response of 1-day-old rat type II cells to surfactant secretagogues. Freshly isolated cells were cultured +/-10(-6) M Dex for 18--20 h after which phosphatidylcholine secretion was measured. Dex slightly decreased the basal secretion rate. However, it significantly increased the response to terbutaline, NECA, ATP and UTP. This effect was dependent on Dex concentration (EC(50)=2-6 x 10(-9) M) and blocked by the glucocorticoid receptor antagonist RU-486. It is unlikely to be due to increased receptor content as Dex had no effect on adenylate cyclase, phospholipase C or phospholipase D activation and the response to cAMP, forskolin and phorbol ester, secretagogues acting downstream from receptors, was also increased by Dex. These data show that Dex acts directly on the type II cell to enhance the response to surfactant secretagogues, that the effect of the hormone is mediated by the glucocorticoid receptor and suggest induction of a common downstream signaling step(s). Regulation of surfactant secretion may be an important function of glucocorticoids in the developing lung.

Inhibition of glycine currents by dextromethorphan in neurones dissociated from the guinea-pig nucleus tractus solitarii.

1. The effect of dextromethorphan (DM) on the current induced by glycine in acutely dissociated nucleus tractus solitarii (NTS) neurones of guinea-pigs was studied by use of the whole-cell patch clamp technique. The effect of DM on gamma-aminobutyric acid (GABA)-induced currents (IGABA) was also examined. 2. DM inhibited 30 microM glycine-induced current (IGly), without affecting the current caused by 30 microM GABA. The action of DM was concentration-dependent, with a maximum effect at 100 microM, and reversible. The half-maximum inhibitory concentration (IC50) of DM was 3.3 microM, about 85 times higher than that of strychnine. 3. DM 3 microM shifted the concentration-response curve for glycine to the right without affecting the maximum value. DM 10 microM shifted the curve even more to the right, although it was not a parallel shift. Strychnine at a concentration of 0.1 microM shifted the curve for glycine in a nearly parallel fashion. 4. The effect of 10 microM DM was slightly weak voltage-dependency, but the lower concentration of DM, 3 microM, inhibited IGly equally at -50 mV and +50 mV. The effect of 3 microM DM on IGly showed no use-dependence. Blockade by strychnine 0.1 microM showed no voltage- or use-dependence. 5. The results indicate that DM inhibits IGly in single neurones of NTS, and further suggest that DM at a low concentration may act on the glycine receptor-ionophore complex, but not on the Cl channel of the complex. However, a relatively high concentration of DM may at least partly affect the Cl- channel of the complex.

Prostaglandin E(2) increases surfactant secretion via the EP(1) receptor in rat alveolar type II cells.

Prostaglandin E(2), the predominant cyclooxygenase metabolite of arachidonic acid in alveolar type II cells, can stimulate pulmonary surfactant secretion. The actions of prostaglandin E(2) are mediated by four prostaglandin E (EP) receptor subtypes designated EP(1), EP(2), EP(3) and EP(4). These subtypes couple to different signal transduction pathways. However, it is not clear which of these subtypes is expressed on type II cells and mediates surfactant secretion. We found that the four subtypes of EP receptors are expressed on the primary cultured alveolar type II cells from adult rats. We also concluded that EP(1) receptor appears to mediate prostaglandin E(2)-induced surfactant secretion through Ca(2+) mobilization.

Differential effect of codeine on coughs caused by mechanical stimulation of two different sites in the airway of guinea pigs.

We studied the difference in the effects of codeine on coughs caused by mechanical stimulation to the larynx and to the bifurcation of the trachea in lightly anaesthetized guinea pigs. Mechanical stimulation to the larynx or the bifurcation of trachea caused a stable cough response. The response was reproducible over 60 min, when stimulation was repeatedly applied at 20-min intervals. No significant difference was found between the amplitudes of the responses to mechanical stimulation of the larynx and of the tracheal bifurcation. Codeine, 10, 20 and 50 mg/kg, dose dependently depressed the coughs caused by larynx stimulation. The antitussive, however, failed to depress the cough caused by stimulation to the tracheal bifurcation, although a large dose, 50 mg/kg, significantly depressed the cough. In capsaicin-treated guinea pigs, codeine at 20 mg/kg significantly depressed the cough caused by stimulation to the tracheal bifurcation. The present results suggest that cough caused by mechanical stimulation to the larynx might be more sensitive to codeine treatment than cough caused by stimulation to the bifurcation of trachea. Furthermore, it is suggested that coughs caused by mechanical stimulation to both sites might consist of at least two components as regards their pharmacological nature.

Both beta 1- and beta 2-adrenoceptors are involved in mediating phosphatidylcholine secretion in rat type II pneumocyte cultures.

A primary culture of rat type II pneumocytes was used for the pharmacological and functional characterization of beta-adrenoceptor subtypes. The beta-adrenoceptor agonists, isoprenaline, dobutamine and procaterol concentration dependently increased the secretion of phosphatidylcholine. These effects were attenuated by propranolol. The effect of dobutamine was attenuated by atenolol, and that of procaterol by ICI 118,551. Isoprenaline-induced secretion was attenuated by the combination of the two blockers but not by each one alone. In conclusion, both beta 1- and beta 2-adrenoceptor subtypes mediate phosphatidylcholine secretion in rat type II pneumocytes.

The influence of neuraminidase treatment on tracheal smooth muscle contraction.

To examine the role of sialic acid in the respiratory tract, the influence of neuraminidase from Clostridium perfringens was investigated on contractions of isolated guinea-pig and rat trachea and on histamine release from guinea-pig lung tissue. Treatment with 2.0 units/ml of neuraminidase at 37 degrees C and pH 7.4 for 30 min caused an approximately 60% removal of total N-acetylneuraminic acid, a representative sialic acid, from muscle from guinea-pig and rat trachea. Neuraminidase concentration dependently induced histamine release from guinea-pig chopped lung tissue, but has no effect on contractions produced by acetylcholine, histamine and 5-hydroxytryptamine. Pretreatment with 2.0 units/ml of neuraminidase inhibited the contraction induced by antigen (ovalbumin) or compound 48/80. These findings suggest, at least in part, that sialic acids sensitive to neuraminidase are involved in the regulation of histamine release but not tracheal contraction.

Adrenomedullin increases phosphatidylcholine secretion in rat type II pneumocytes.

Adrenomedullin, a novel hypotensive peptide, has been reported to be produced in the lung as well as in the adrenal medulla. However, the effect of adrenomedullin on lung function is still poorly understood. In this study, we detected the expression of both adrenomedullin mRNA and putative adrenomedullin receptor mRNA in primary cultures of rat type II pneumocytes. Adrenomedullin increased the secretion of phosphatidylcholine, the predominant component of pulmonary surfactant, by type II pneumocytes. The increase was partly inhibited by pretreatment with the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP-(8-37). Furthermore, the increased phosphatidylcholine secretion was significantly inhibited by several protein kinase C inhibitors, such as sphingosine, 2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]3-(1H-indol-3-yl) maleimide (Gö6983), 3-[1-(3-amidinothio)-propyl-1H-indoyl-3-yl]3-(1-methyl-1H-++ +indoyl-3-yl ) maleimide methane sulfonate (Ro-31-8220), and staurosporine. Our results suggest that adrenomedullin can be considered a candidate autocrine modulator of surfactant secretion in type II pneumocytes.

Stimulation of pulmonary surfactant secretion by activating neutrophils in rat type II pneumocytes culture.

The influence of activating neutrophils on the secretion of phosphatidylcholine (PC), the predominant component of pulmonary surfactant, was examined using primary culture of rat type II pneumocytes. Simultaneous addition of neutrophils and opsonized zymosan, but not neutrophils or opsonized zymosan alone, to type II pneumocytes caused a significant increase in PC secretion without affecting the release of lactate dehydrogenase, a marker of cytotoxicity. The increase in PC secretion was dependent on the number of activating neutrophils. In addition, pretreatment of culture with the combination of superoxide dismutase and catalase inhibited the increase in PC secretion. These findings indicate that activating neutrophils stimulate the secretion of pulmonary surfactant and that the stimulation is mediated by oxygen radicals.

Interaction between beta-adrenergic signaling and protein kinase C increases cytoplasmic Ca2+ in alveolar type II cells.

The interaction between beta-adrenergic signaling and the activation of protein kinase C in alveolar type II cell plays an important role in the regulation of surfactant secretion because the combined application of beta-adrenergic agonist with protein kinase C activator to the cells stimulates the secretion synergistically. However, the mechanisms underlying the interaction are not clear. In the present study, we examined the combined effect of terbutaline with phorbol 12-myristate 13-acetate (PMA) on cytoplasmic free Ca2+ concentration ([Ca2+]i) in rat alveolar type II cells. The combined application of terbutaline with PMA to the cells rapidly increased [Ca2+]i, although neither of them affected it by itself. Similar increases of [Ca2+]i were observed in other combinations, such as terbutaline with 1-oleoyl-2-acetyl-sn-glycerol, and forskolin with PMA. Either the removal of extracellular Ca2+ or the addition of Co2+ remarkably suppressed the increase of [Ca2+]i induced by the combination of terbutaline with PMA. In addition, Co2+ inhibited the phosphatidylcholine secretion induced by the combination of terbutaline and PMA. These results suggested that the [Ca2+]i increased as a result of the interaction between formation of cyclic AMP and activation of protein kinase C in alveolar type II cells, and that the increase in [Ca2+]i was mediated by the Ca2+ influx through the plasma membrane. This mechanism to modulate [Ca2+]i may play a role in the regulation of surfactant secretion by alveolar type II cells.

Carbenoxolone, a new inducer of heat shock protein 70.

Heat shock protein 70 (Hsp70) is capable of protecting cells, tissues, organs, and animals from a subsequent, normally lethal heating, as well as from numerous disease states. Therefore, it would be of great therapeutic benefit to discover compounds that are clinically safe yet able to induce Hsp70 in patients. Carbenoxolone, an antiulcer drug, protects gastric mucosal cells against irritants in vivo and in vitro. We assessed here whether carbenoxolone induces Hsp70 expression in human cell lines. We found that carbenoxolone increased the expression of Hsp70 protein and mRNA, and Hsp70 promoter activity.

Mucolytic and antitussive effects of erdosteine.

To investigate the influence of erdosteine, a new homocysteine-derived expectorant, on airway clearance we studied the effects of the drug on the viscosity of mucin, on the mucociliary transport rate in quails, on airway secretion in rats and on the cough reflex in guinea-pigs. The active metabolite of erdosteine, M1 (10 microM to 1 mM), significantly reduced the viscosity of porcine stomach mucin. Erdosteine by itself did not reduce viscosity. Erdosteine significantly promoted mucociliary transport in quails and increased airway secretion in rats. The effect was still apparent 24h after administration. Erdosteine significantly suppressed citric acid-induced cough reflexes in guinea-pigs but did not suppress mechanical stimuli-induced cough reflexes. Erdosteine suppressed the reduction of the recovery volume of bronchoalveolar lavage fluid and albumin leakage into the fluid in citric acid-exposed guinea-pigs. These results indicate that erdosteine removes sputum by reducing its viscosity, and by promoting mucociliary transport and sustained enhancement of airway secretion. It also suppressed the chemical stimulation-induced cough reflex and plasma leakage into the airway. These results suggest that erdosteine is an excellent expectorant with several modes of action.

Pyridine derivatives stimulate phosphatidylcholine secretion in primary cultures of rat type II pneumocytes.

We have examined the effects of pyridine derivatives on phosphatidylcholine secretion in primary cultures of rat type II pneumocytes. Of 12 pyridine derivatives, 4-aminopyridine, 4-dimethylaminopyridine and 4-pyrolidinopyridine had a stimulatory effect on phosphatidylcholine secretion, whereas other derivatives had little effect. The stimulatory effect of 4-aminopyridine was concentration- and time-dependent, and was inhibited by the acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (3 microM), an intracellular Ca2+ chelator. In addition, the stimulatory effect of 4-aminopyridine was suppressed by W-7(N-(6-aminohexyl)-5-chloro-1-napthalene-sulphonamide)(10 microM), a calmodulin inhibitor, and sphingosine (10 microM) and staurosporine (0-1 microM), protein kinase C inhibitors. These results indicate that several pyridine derivatives stimulate phosphatidylcholine secretion in type II pneumocytes.

Inhibition of glycine-induced current by morphine in nucleus tractus solitarii neurones of guinea pigs.

We studied the effect of morphine on the current induced by glycine in acutely dissociated nucleus tractus solitarii (NTS) neurones of guinea pigs, by use of the whole-cell patch clamp technique. Morphine inhibited 30 microM glycine-induced current (Igly), without affecting the current caused by 30 microM GABA. The effect of morphine was concentration-dependent, with a maximal effect at 1 mM, and reversible. The half-maximum inhibitory concentration of morphine was 30 microM. The effect of morphine was not depressed by naloxone, an opioid antagonist. Furthermore, the effect was not substantially affected by methiothepin, a 5-HT1 antagonist, ketanserin, a 5-HT2 antagonist and MDL-72222, a 5-HT3 antagonist. Morphine at 30 microM shifted the concentration-response curve for Igly to the right without affecting the maximum value. The effect of morphine on Igly showed no use-dependence. The results indicate that morphine inhibits Igly in the NTS neurones, and further suggest that morphine at the concentration used may act on the glycine receptor-ionophore complex, but not on the Cl-channel of the complex.

Nitric oxide/soluble guanylyl cyclase signaling mediates depolarization-induced protection of rat mesencephalic dopaminergic neurons from MPP⁺ cytotoxicity.

Neuronal electrical activity has been known to affect the viability of neurons in the central nervous system. Here we show that long-lasting membrane depolarization induced by elevated extracellular K(+) recruits nitric oxide (NO)/soluble guanylyl cyclase/protein kinase G signaling pathway, induces 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP)-mediated protein S-guanylation, and confers dopaminergic neuroprotection. Treatment of primary mesencephalic cell cultures with 1-methyl-4-phenylpyridinium (MPP(+)) for 72 h decreased the number of dopaminergic neurons, whereas the cell loss was markedly inhibited by elevated extracellular concentration of K(+) (+40 mM). The neuroprotective effect of elevated extracellular K(+) was significantly attenuated by tetrodotoxin (a Na(+) channel blocker), amlodipine (a voltage-dependent Ca(2+) channel blocker), N(ω)-nitro-l-arginine methyl ester (l-NAME) (a nitric oxide synthase inhibitor), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (a soluble guanylyl cyclase inhibitor), and KT5823 or Rp-8-bromo-ß-phenyl-1,N(2)-ethenoguanosine 3',5'-cyclic monophosphorothioate (Rp-8-Br-PET-cGMPS) (protein kinase G inhibitors). Elevated extracellular K(+) increased 8-nitro-cGMP production resulting in the induction of protein S-guanylation in cells in mesencephalic cultures including dopaminergic neurons. In addition, exogenous application of 8-nitro-cGMP protected dopaminergic neurons from MPP(+) cytotoxicity, which was prevented by zinc protoporphyrin IX, an inhibitor of heme oxygenase-1 (HO-1). Zinc protoporphyrin IX also inhibited the neuroprotective effect of elevated extracellular K(+). On the other hand, KT5823 or Rp-8-Br-PET-cGMPS did not inhibit the induction of HO-1 protein expression by 8-nitro-cGMP, although these protein kinase G inhibitors abrogated the neuroprotective effect of 8-nitro-cGMP. These results suggest that protein S-guanylation (leading to HO-1 induction) as well as canonical protein kinase G signaling pathway plays an important role in NO-mediated, activity-dependent dopaminergic neuroprotection.

Caffeic acid phenethyl ester protects nigral dopaminergic neurons via dual mechanisms involving haem oxygenase-1 and brain-derived neurotrophic factor.

BACKGROUND AND PURPOSE: Caffeic acid phenethyl ester (CAPE) is a component of honey bee propolis that can induce expression of haem oxygenase-1 (HO-1). Because HO-1 induction has been suggested to protect dopaminergic neurons in the substantia nigra, we examined the effect of CAPE in experimental models of dopaminergic neurodegeneration. EXPERIMENTAL APPROACH: Neuroprotective effect of CAPE was investigated in rat organotypic midbrain slice cultures and in vivo, using a mouse model of dopaminergic neurodegeneration induced by intranigral injection of LPS and intrastriatal injection of 6-hydroxydopamine. KEY RESULTS: CAPE protected dopaminergic neurons in slice cultures from IFN-γ/LPS-induced injury. The effect of CAPE was inhibited by zinc protoporphyrin IX, an HO-1 inhibitor, and by neutralizing antibody against brain-derived neurotrophic factor (BDNF). A p38 MAPK inhibitor SB203580 prevented activation of NF-E2-related factor 2, attenuated increased expression of HO-1 and BDNF, and blocked the neuroprotective actions of CAPE. In the LPS-injected mouse model, daily intraperitoneal administration of CAPE protected dopaminergic neurons, up-regulated HO-1 and BDNF, and reduced the increase of activated microglia/macrophages. Neuroprotective effects of CAPE against LPS-induced injury was prevented by zinc protoporphyrin IX or anti-BDNF antibody. CAPE protected dopaminergic neurons and alleviated methamphetamine-induced rotational behaviour also in 6-hydroxydopamine hemiparkinsonian mice. CONCLUSION AND IMPLICATIONS: CAPE is a novel type of neuroprotective agent whose actions are mediated by both HO-1 and BDNF. These findings may provide novel clues to develop neuroprotective agents for treatment of neurodegenerative disorders.

α7 Nicotinic acetylcholine receptor agonist attenuates neuropathological changes associated with intracerebral hemorrhage in mice.

We have demonstrated previously that nicotine affords neuroprotective and anti-inflammatory effects against intracerebral hemorrhage (ICH)-associated neuropathological changes. The present study was undertaken to clarify whether subtype-specific agonists of nicotinic acetylcholine receptors (nAChRs) could preserve tissue integrity in mouse ICH model in vivo. ICH was induced by unilateral injection of collagenase into the striatum of male C57BL/6 mice. Daily intraperitoneal injection of α7 nAChR agonist PNU-282987 (3-10mg/kg) for 3 days, starting from 3h after induction of ICH, significantly increased the number of surviving neurons in the central and the peripheral regions of hematoma at 3 days after ICH. In contrast, α4ß2 nAChR agonist RJR-2403 (2-10 mg/kg) given in the same regimen showed no significant effect. PNU-282987 and RJR-2403 did not affect either the size of hemorrhage or the extent of brain edema associated with ICH. PNU-282987 decreased the number of activated microglia/macrophages accumulating in the perihematoma region at 3 days after ICH, in a dose-dependent manner. On the other hand, the number of microglia/macrophages in the central region of hematoma at early phase of pathology (6 h after ICH) was increased by 10mg/kg PNU-282987. These results suggest that α7 nAChR agonist can provide neuroprotective effect on ICH-induced injury, independently of its anti-inflammatory actions.

Orexin neurons in hypothalamic slice cultures are vulnerable to endoplasmic reticulum stress.

Narcolepsy results from disruption of orexin neurons in the hypothalamus that play a key role in maintenance of the arousal state. Underlying mechanisms leading to selective loss of orexin neurons remain unknown. On the other hand, endoplasmic reticulum stress, namely, conditions associated with impairment of endoplasmic reticulum functions such as proper folding and sorting of newly synthesized proteins, is implicated in pathogenesis of several types of neurodegenerative disorders. Here we found that application of endoplasmic reticulum stress inducers such as tunicamycin (that prevents protein N-glycosylation) and thapsigargin (that inhibits Ca²âº-ATPase) to organotypic slice cultures of the hypothalamus caused preferential loss of orexin-immunoreactive neurons, as compared to melanin-concentrating hormone- or calcitonin gene-related peptide-immunoreactive neurons. The decrease in orexin-immunoreactive neurons at early time points (6-24 h) was not accompanied by induction of cell death as indicated by the absence of caspase-3 activation and no significant change in the number of NeuN-positive cells, whereas sustained treatment with tunicamycin for 72 h induced cell death. At 24-h treatment, tunicamycin and thapsigargin did not decrease expression of prepro-orexin mRNA, suggesting that post-transcriptional mechanisms were responsible for depletion of orexin peptides. In addition, inhibition of axonal transport by colchicine and inhibition of proteasomal activity by MG132 significantly prevented the decrease in orexin immunoreactivity by tunicamycin. Comparative examinations of expression of unfolded protein response-related proteins revealed that C/EBP-homologous protein (a transcription factor that promotes induction of apoptosis) as well as phosphorylated form of RNA-dependent protein kinase-like endoplasmic reticulum kinase (a protein kinase that mediates inhibition of protein translation) was expressed more prominently in orexin neurons than in melanin-concentrating hormone neurons, in response to tunicamycin. These results indicate that orexin neurons are particularly sensitive to endoplasmic reticulum stress, which may be relevant to pathogenic events in narcolepsy.

Quinolinate induces selective loss of melanin-concentrating hormone neurons, rather than orexin neurons, in the hypothalamus of mice and young rats.

Orexins are neuropeptides produced in the lateral hypothalamus and implicated in regulation of sleep-wake cycle. Selective loss of orexin neurons is found in the brain of patients with narcolepsy, but the mechanisms of this pathological change are unclear. A previous study showed that excessive stimulation of N-methyl-d-aspartate (NMDA) receptors by quinolinic acid (QA) caused selective loss of orexin neurons in rat hypothalamic slice culture. Here we examined QA toxicity on orexin neurons and melanin-concentrating hormone (MCH) neurons in vivo. Contrary to the expectation, injection of QA (60 and 120 nmol) into the lateral hypothalamus of male C57BL/6 mice caused selective loss of MCH neurons rather than orexin neurons, and this toxicity of QA was attenuated by MK-801, an NMDA receptor antagonist. Selective loss of MCH neurons with preserved orexin neurons was observed even when GABA(A) receptor antagonists such as bicuculline and picrotoxin were injected with QA. A significant decrease in the number of orexin neurons was induced when QA injection was performed in the dark phase of diurnal cycle, but the degree of the decrease was still lower than that in the number of MCH neurons. Finally, QA (60 nmol) induced selective loss of MCH neurons also in young rats at 3-4 weeks of age. These results do not support the hypothesis that acute excitotoxicity mediated by NMDA receptors is responsible for the pathogenesis of narcolepsy.

Induction of EMT-like phenotypes by an active metabolite of leflunomide and its contribution to pulmonary fibrosis.

Drug-induced interstitial lung disease (ILD), particularly pulmonary fibrosis, is a serious clinical concern and myofibroblasts have been suggested to have a major role, with it recently being revealed that some of these myofibroblasts are derived from lung epithelial cells through epithelial-mesenchymal transition (EMT). In this study, we examined the EMT-inducing abilities of drugs known to induce ILD clinically. EMT-like phenotypes were induced by A771726, an active metabolite of leflunomide having an inhibitory effect on dihydroorotate dehydrogenase (DHODH). Smad-interacting protein 1 (a transcription factor regulating EMT) and the Notch-signaling pathway but not transforming growth factor-ß was shown to be involved in A771726-induced EMT-like phenotypes. When the cultures were supplemented with exogenous uridine, the A771726-induced EMT-like phenotypes and activation of the Notch-signaling pathway disappeared. Similarly, an A771726 analog without inhibitory activity on DHODH produced no induction, suggesting that this process is mediated through the inhibition of DHODH. In vivo, administration of leflunomide stimulated bleomycin-induced EMT-like phenomenon in pulmonary tissue, and exacerbated bleomycin-induced pulmonary fibrosis, both of which were suppressed by coadministration of uridine. Taken together, these findings suggest that leflunomide-dependent exacerbation of bleomycin-induced pulmonary fibrosis is mediated by stimulation of EMT of lung epithelial cells, providing the first evidence that drug-induced pulmonary fibrosis involves EMT of these cells.