Characteristics of EPI-hNE4 aerosol: a new elastase inhibitor for treatment of cystic fibrosis.

The purpose of this study was to define nebulization conditions providing delivery of aerosols of EPI-hNE4, an inhibitor of human neutrophil elastase (HNE). EPI-hNE4 was nebulized with Pari LC Star and tested at three concentrations (2.5, 5, and 10 mg/mL). The inhaled mass was measured over 15 min. Particle size distribution was measured by cascade impaction. The effect was also tested of mixing EPI-hNE4 with a (99m)Tc human serum albumin (HSA) tracer on the aerodynamic properties of the aerosol. The inhibitory activity of EPI-hNE4 after nebulization was assessed on purified HNE. The inhaled mass was 32.3 +/- 3.5% (mean +/- SD) after 10 min and 44.2 +/- 3.8% (mean +/- SD) after 15 min. Mass median aerodynamic diameter ranged between 1.2 and 1.8 microm. The (99m)Tc HSA EPI-hNE4 aerosol was similar in terms of particle size distribution (y = 1.0338x - 0.003, r = 0.83). (99m)Tc activity was predictive of EPI-hNE4 mass distribution (y = 1.0278x - 1.6991, r = 0.89). The inhibitory capacity of aerosolized samples remained unchanged after up to 10 min of nebulization. EPI-hNE4 can be nebulized efficiently without decrease in its activity. Mixing this inhibitor with (99m)Tc HSA should allow quantification of its deposition in CF patients.

Group I metabotropic glutamate receptor inhibition selectively blocks a prolonged Ca(2+) elevation associated with age-dependent excitotoxicity.

It has been recognized for some years that a prolonged Ca(2+) elevation that is predictive of impending cell death develops in cultured neurons following excitotoxic insult. In addition, neurons exhibit enhanced sensitivity to excitotoxic insult with increasing age in culture. However, little is known about the processes that selectively regulate the post-insult Ca(2+) elevation and therefore, it remains unclear whether it is associated specifically with age-dependent toxicity.Here, we tested the hypothesis that a group I metabotropic glutamate receptor antagonist selectively modulates the prolonged Ca(2+) elevation in direct association with its protective effects against excitotoxicity. Rat hippocampal cultures of two ages (8-9 and 21-28 days in vitro) were exposed to a 5-min glutamate insult (400 microM in younger and 10 microM in older cultures) sufficient to kill >50% of the neurons, and were treated with vehicle or the specific group I metabotropic glutamate receptor antagonist 1-aminoindan-1,5-dicarboxylic acid (AIDA; 1 mM), throughout and following the insult. Neuronal survival was quantified 24 h after insult. In parallel studies, neurons of similar age in culture were imaged ratiometrically with a confocal microscope during and for 60 min after the glutamate insult. A large post-insult Ca(2+) elevation was present in older but not most younger neurons. The N-methyl-D-aspartate receptor antagonist, MK-801, blocked the Ca(2+) elevation both during and following the insult. In contrast, AIDA blocked only the post-insult prolonged Ca(2+) elevation in older neurons. Moreover, AIDA was neuroprotective in older but not younger cultures. From these results we suggest that the post-insult Ca(2+) elevation is regulated differently from the Ca(2+) elevation during glutamate insult and is modulated by group I metabotropic glutamate receptors. Further, the prolonged Ca(2+) elevation appears to be directly linked to an age-dependent component of vulnerability.

Metabotropic glutamate receptors stimulate phospholipase D via different pathways in the adult and neonate rat hippocampus.

In order to characterize the ontogenetic profile of metabotropic glutamate (mGlu) receptors coupled to phospholipase D (PLD) we examined the effects of selected mGlu agents on PLD activity in immature and adult rat hippocampus. The group I mGlu receptor agonist 3,5-dihydroxyphenylglycine stimulated PLD in immature tissue, but reduced the PLD response evoked by the nonselective mGlu receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate [(1S,3R)-ACPD] in adult hippocampus. (2R,1'S,2'R,3'S)-2-(2'-Carboxy-3'-phenylcyclopropyl) glycine (PCCG-13), a recently characterized selective antagonist of PLD-coupled mGlu receptors, displayed a much greater activity in reducing the PLD response to (1S,3R)-ACPD in adult than in neonate hippocampus. Our results lend support to the hypothesis that glutamatergic activation of PLD in the rat hippocampus is developmentally regulated.

Pneumorachis and pneumomediastinum caused by repeated Müller's maneuvers: complications of marijuana smoking.

Pneumomediastinum may occur during marijuana inhalation but only rarely has pneumorachis (epidural pneumatosis or aerorachia) been reported. The usual mechanisms that produce pneumomediastinum include severe acute asthma, toxic-induced bronchial hyperreactivity, and barotrauma caused by Valsalva's maneuver (expiration through resistance). We report a case in which barotrauma resulted from repeated deep inspiration through a device with airflow resistance equivalent to Müller's maneuver. Inspiration occurred through a homemade apparatus resembling a narrow outlet bong with 2 piled compartments. Pneumomediastinum combined with subcutaneous emphysema and pneumorachis occurred, without identified pneumothorax. There were no neurologic complications. Because of the absence of bronchospasm, expiration either through the apparatus or actively against a closed glottis, or apnea, this phenomenon is likely a result of repeated Müller's maneuvers. Successive inhalation through resistance could have resulted in extreme negative intrathoracic pressure, which would have caused a transmural pressure gradient inducing barotrauma and release of extrarespiratory air. High-concentration oxygen therapy to achieve nitrogen washout was used.

[Beginning rheumatoid arthritis revealed during bronchiectasis surinfections. Value of cyclic antibiotherapy?]. / Polyarthrite rhumatoïde débutante révélée au décours de surinfections de bronchectasies. Place d'une antbiothérapie cyclique?

Incidence of symptomatic bronchiectasis (BR) occurs in around 2% in patients with late rheumatoid arthritis (RA). Its seems that the association BR-RA could be a worsening factor for outcome of RA patients. A 58-year-old woman without dry syndrome, suffering from bronchial purulence over one year was admitted to the Department of Pneumology for hemoptysis and arthritis (knees, ankles, and wrists). Three prior episodes of inflammatory articular pain had occurred after transient bronchial purulence or pneumonitis. CT-scan showed bilateral bronchiectasis. Diagnosis of early RA was proved after the third episode of bronchial purulence related to a strain of Haemophilus influenzae. A strain of Coxiella burnetii was probably responsible for one of the three bronchial surinfections. Latex and Waaler Rose tests were transiently positive during the first episode, and became positive after the third one. At that time, RA was relevant in view of ARA criteria. Cyclic prophylactic antibiotic regimens could be proposed to patients suffering from RA-BR association, in contrast to the cases of patients with isolated BR. This approach could prevent destabilization of RA and reinforce of anti-rheumatic therapy. Activation and release of cytokines (NFk-B, TNF-alpha), and/or bacterial epitopes seems to be directly responsible for the articular destabilization.

Presynaptic kynurenate-sensitive receptors inhibit glutamate release.

Kynurenic acid is a tryptophan metabolite provided with antagonist activity on ionotropic glutamate and alpha7 nicotinic acetylcholine receptors. We noticed that in rats with a dialysis probe placed in the head of their caudate nuclei, local administration of kynurenic acid (30-100 nM) significantly reduced glutamate output. Qualitatively and quantitatively similar effects were observed after systemic administration of kynurenine hydroxylase inhibitors, a procedure able to increase brain kynurenate concentrations. Interestingly, in microdialysis studies, methyllycaconitine (0.3-10 nM), a selective alpha7 nicotinic receptor antagonist, also reduced glutamate output. In isolated superfused striatal synaptosomes, kynurenic acid (100 nM), but not methyllycaconitine, inhibited the depolarization (KCl 12.5 mM)-induced release of transmitter or previously taken-up [3H]-D-aspartate. This inhibition was not modified by glycine, N-methyl-D-aspartate or subtype-selective kainate receptor agents, while CNQX or DNQX (10 microM), two AMPA and kainate receptor antagonists, reduced kynurenic acid effects. Low concentrations of kynurenic acid, however, did not modify [3H]-kainate (high and low affinity) or [3H]-AMPA binding to rat brain membranes. Finally, because metabotropic glutamate (mGlu) receptors modulate transmitter release in striatal preparations, we evaluated, with negative results, kynurenic acid (1-100 nM) effects in cells transfected with mGlu1, mGlu2, mGlu4 or mGlu5 receptors. In conclusion, our data show that kynurenate-induced inhibition of glutamate release is not mediated by glutamate receptors. Nicotinic acetylcholine receptors, however, may contribute to the inhibitory effects of kynurenate found in microdialysis studies, but not in those found in isolated synaptosomes.

Activation of type 5 metabotropic glutamate receptors enhances NMDA responses in mice cortical wedges.

1. We measured the effects of agonists and antagonists of metabotropic glutamate (mGlu) receptors (types 1 and 5) on NMDA-induced depolarization of mouse cortical wedges in order to characterize the mGlu receptor type responsible for modulating NMDA responses. We also characterized a number of mGlu receptor agents by measuring [3H]-inositol phosphate (IP) formation in cortical slices and in BHK cells expressing either mGlu 1 or mGlu 5 receptors. 2. (S)-3,5-dihydroxyphenylglycine (DHPG), an agonist of both mGlu 1 and mGlu 5 receptors, at concentrations ranging from 1-10 microM, enhanced up to 105+/-15% the NMDA-induced depolarization. Larger concentrations (100-300 microM) of the compound were inactive in this test. When evaluated on [3H]-IP synthesis in cortical slices or in cells expressing either mGlu 1 or mGlu 5 receptors, DHPG responses (1-300 microM) increased in a concentration-dependent manner. 3. (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) and (S:)-(+)-2-(3'-carboxybicyclo[1.1.1]pentyl)-glycine (CBPG), had partial agonist activity on mGlu 5 receptors, with maximal effects reaching approximately 50% that of the full agonists. These compounds, however, enhanced NMDA-evoked currents with maximal effects not different from those induced by DHPG. Thus the enhancement of [3H]-IP synthesis and the potentiation of NMDA currents were not directly related. 4. 2-methyl-6-(phenylethynyl)-pyridine (MPEP, 1-10 microM), a selective mGlu 5 receptor antagonist, reduced DHPG effects on NMDA currents. 7-(hydroxyimino)cyclopropan[b]-chromen-1a-carboxylic acid ethylester (CPCCOEt, 30 microM), a preferential mGlu 1 receptor antagonist, did not reduce NMDA currents. 5. These results show that mGlu 5 receptor agonists enhance while mGlu 5 receptor antagonists reduce NMDA currents. Thus the use of mGlu 5 receptor agents may be suggested in a number of pathologies related to altered NMDA receptor function.

Biochemical and electrophysiological studies on (S)-(+)-2-(3'-carboxybicyclo(1.1.1)pentyl)-glycine (CBPG), a novel mGlu5 receptor agonist endowed with mGlu1 receptor antagonist activity.

The pharmacological profile of (S)-(+)-2-(3'-carboxybicyclo[1.1.1]pentyl)-glycine (CBPG) and of other group 1 metabotropic glutamate (mGlu) receptor agents were studied in BHK cells transfected with mGlu receptor subtypes or in native receptors in brain slices by measuring second messenger responses. The mGlu receptor-mediated changes in the electrophysiological properties of CA1 pyramidal cells of the hippocampus were also evaluated. In mGlu5a receptor transfected cells, CBPG behaved as a partial agonist, while in mGlu1alpha receptor transfected cells, it behaved as a glutamate antagonist. No effect was found on cAMP formation in cells transfected with mGlu2 receptors or mGlu4 receptors. In brain slices, CBPG neither affected phospholipase D-coupled glutamate receptors nor did it modify the responses to ionotropic receptor stimulation (at concentrations up to 1 mM). When tested in CA1 pyramidal cells of the hippocampus, CBPG (50-100 microM) caused depolarization, increased cell input resistance, and decreased action potential frequency adaptation and afterhyperpolarization. DHPG (3-100 microM), an agonist of both mGlu1 and mGlu5 receptors, and CHPG (1000 microM), a low affinity mGlu5 agonist, produced qualitatively similar effects. The actions of CBPG or CHPG were not modified by AIDA (300 microM), a selective mGlu1 receptor antagonist. Our results suggest that CBPG could be a useful tool for discriminating between mGlu1 receptor and mGlu5 receptor effects and that mGlu5 receptors are the receptors which are mainly responsible for the direct excitatory effects of mGlu receptor agonists on CA1 pyramidal cells.

Antagonist pharmacology of metabotropic glutamate receptors coupled to phospholipase D activation in adult rat hippocampus: focus on (2R,1'S,2'R,3'S)-2-(2'-carboxy-3'-phenylcyclopropyl)glycine versus 3, 5-dihydroxyphenylglycine.

Metabotropic glutamate (mGlu) receptors coupled to phospholipase D (PLD) appear to be distinct from any known mGlu receptor subtype linked to phospholipase C or adenylyl cyclase. The availability of antagonists is necessary for understanding the role of these receptors in the central nervous system, but selective ligands have not yet been identified. In a previous report, we observed that 3, 5-dihydroxyphenylglycine (3,5-DHPG) inhibits the PLD response induced by (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate in adult rat hippocampal slices. We now show that the antagonist action of 3, 5-DHPG (IC50 = 70 microM) was noncompetitive in nature and nonselective, because the drug was also able to reduce PLD activation elicited by 100 microM norepinephrine and 1 mM histamine. In the search for a selective and more potent antagonist, we examined the effects of sixteen stereoisomers of 2-(2'-carboxy-3'-phenylcyclopropyl)glycine (PCCG) on the PLD-specific transphosphatidylation reaction resulting in the formation of [3H]phosphatidylethanol. The (2R,1'S,2'R,3'S)-PCCG stereoisomer (PCCG-13) antagonized the formation of [3H]phosphatidylethanol induced by 100 microM (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylate in a dose-dependent manner and with a much lower IC50 value (25 nM) compared with 3,5-DHPG. In addition, increasing concentrations of PCCG-13 were able to shift to the right the agonist dose-response curve but had no effect when tested on other receptors coupled to PLD. The potent, selective, and competitive antagonist PCCG-13 may represent an important tool for elucidating the role of PLD-coupled mGlu receptors in adult hippocampus.

Type 2 metabotropic glutamate (mGlu) receptors tonically inhibit transmitter release in rat caudate nucleus: in vivo studies with (2S,1'S,2'S,3'R)-2-(2'-carboxy-3'-phenylcyclopropyl)glycine, a new potent and selective antagonist.

Anatomical, biochemical and electrophysiological studies have previously shown that cortico-striatal terminals contain abundant presynaptic group 2 metabotropic glutamate (mGlu) receptors. Using brain slices we have previously shown that these receptors inhibit depolarization-induced transmitter release. Using microdialysis in freely moving rats, we now report the effects of group 2 mGlu receptor agonists and antagonists on glutamate concentration in the caudate extracellular fluid. A mild decrease (20-30%) in glutamate concentration in caudate dialysates was observed when 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid or (2S,3S,4S)-alpha-carboxycyclopropyl-glycine (L-CCG-1), mGlu receptor agonists, was locally administered. On the contrary, alpha-methyl-4-carboxyphenylglycine, an antagonist of type 1 and type 2 mGlu receptors, increased the glutamate concentration in dialysates by up to 3.5-fold, and its effects were prevented by the simultaneous administration of L-CCG-1, a preferential type 2 mGlu receptor agonist. A significant increase of glutamate output in striatal dialysate was also found after local administration of (2S,1'S,2'S,3'R)-2-(2'-carboxy-3'-phenylcyclopropyl)glycine, another structurally unrelated, relatively selective and potent type 2 mGlu receptor antagonist. The results suggest that type 2 mGlu receptors tonically inhibit transmitter release from cortico-striatal terminals. Since the cortico-striatal pathway profoundly affects the function of a large percentage of caudate neurons, it is reasonable to predict that the use of selective type 2 mGlu receptor agents will be helpful for scientific and therapeutic studies on the physiopathology of basal ganglion disorders.

Pharmacological characterization of 1-aminoindan-1,5-dicarboxylic acid, a potent mGluR1 antagonist.

We examined the pharmacological profile of 1-aminoindan-1,5-dicarboxylic acid (AIDA), a rigid (carboxyphenyl)glycine derivative acting on metabotropic glutamate receptors (mGluRs). In cells transfected with mGluR1a, AIDA competitively antagonized the stimulatory responses of glutamate and (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD] on phosphoinositide hydrolysis (pA2 = 4.21). In cells transfected with mGluR5a, AIDA displayed a much weaker antagonist effect. In transfected cells expressing mGluR2, AIDA (< or = 1 mM) did not affect the inhibition of forskolin-stimulated adenylate cyclase activity induced by (1S,3R)-ACPD, but at large concentrations, it displayed a modest agonist activity. In rat hippocampal or striatal slices, AIDA (0.1-1 mM) reduced the effects of (1S,3R)-ACPD on phospholipase C but not on adenylate cyclase responses, whereas (+)-alpha-methyl-4-carboxyphenylglycine (0.3-1 mM) was an antagonist on both transduction systems. In addition, AIDA (0.3-1 mM) had no effect on mGluRs coupled to phospholipase D, whereas (+)-alpha-methyl-4-carboxy-phenylglycine (0.5-1 mM) acted as an agonist with low intrinsic activity. In rat cortical slices, AIDA antagonized the stimulatory (mGluR1-mediated) effect of (1S,3R)-ACPD on the depolarization-induced outflow of D-[3H]aspartate, disclosing an inhibitory effect ascribable to (1S,3R)-ACPD activating mGluR2 and/or mGluR4. Finally, mice treated with AIDA (0.1-10 nmol i.c.v.) had an increased pain threshold and difficulties in initiating a normal ambulatory behavior. Taken together, these data suggest that AIDA is a potent, selective and competitive mGluR1 a antagonist.

Farnesyl pyrophosphate synthase from white lupin: molecular cloning, expression, and purification of the expressed protein.

Plants produce a variety of sesquiterpenoid compounds with diverse biological functions, whose synthesis is initiated by farnesyl pyrophosphate synthase [EC 2.5.1.1, EC 2.5.1.10]. The lack of availability of molecular tools to analyze this enzyme has, thus far, prevented the study of its expression and regulation in plants. A DNA fragment corresponding to a portion of the farnesyl pyrophosphate synthase gene was amplified by the polymerase chain reaction using was amplified by the polymerase chain reaction using degenerate primers designed from two highly conserved domains (FLV(A/L)DD(I/M)MD and FQIQDDYLD) found in eukaryotic farnesyl pyrophosphate synthase sequences. A clone, pS19, of a 438-bp PCR fragment was used to screen a white lupin root cDNA library. Two full-length cDNA clones (pFPS1 and pFPS2) were isolated and sequenced, and one of them (pFPS2) was expressed in a bacterial system and the enzyme protein encoded by the clone was purified. The 1345-bp insert of pFPS2 contains a 1026-bp open reading frame, encoding a 342-amino-acid peptide with a calculated molecular mass of 39,310 Da. The deduced amino acid sequence of lupin farnesyl pyrophosphate synthase pFPS2 shares 90 and 79% identity with those from Lupinus albus (pFPS1) and Arabidopsis thaliana, respectively, 51% with the yeast enzyme, and 44% identity with those from rat and human. The overexpressed protein, which was purified to near homogeneity, displayed both dimethylallyl transferase and geranyl transferase activities. Polyclonal antibodies raised against the purified protein immunorecognized a ca 39-kDa protein in lupin root extracts.

Oxidative phosphorylation by mitochondria extracted from dry sunflower seeds.

The role of mitochondria in the phosphorylation of ADP to ATP in the early steps of seed germination has been studied. Mitochondria were extracted from dry sunflower (Helianthus annuus) seeds. Adenylate kinase-dependent ATP synthesis was inhibited by p(1),p(5)-di(adenosine-5')pentaphosphate. Synthesis of ATP was observed with the different substrates: citrate, alpha-ketoglutarate, succinate, malate, pyruvate or NADH. This synthesis was activated by cytochrome c, and inhibited by cyanide, oligomycin, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, and carboxyatractyloside. The ATP/O values with succinate were 0.85 and 1.2 in the absence or presence, respectively, of cytochrome c. Electron micrographs showed that mitochondria of dry tissues have different structures when observed in situ or in vitro after aqueous extraction, suggesting that profound changes occurred after the contact with the aqueous medium. These results confirm previous data obtained in vivo showing that mitochondria present in dry seeds are able to synthesize ATP as soon as the seeds are rehydrated.