What the pulmonary specialist should know about the new inhalation therapies.

A collaboration of multidisciplinary experts on the delivery of pharmaceutical aerosols was facilitated by the European Respiratory Society (ERS) and the International Society for Aerosols in Medicine (ISAM), in order to draw up a consensus statement with clear, up-to-date recommendations that enable the pulmonary physician to choose the type of aerosol delivery device that is most suitable for their patient. The focus of the consensus statement is the patient-use aspect of the aerosol delivery devices that are currently available. The subject was divided into different topics, which were in turn assigned to at least two experts. The authors searched the literature according to their own strategies, with no central literature review being performed. To achieve consensus, draft reports and recommendations were reviewed and voted on by the entire panel. Specific recommendations for use of the devices can be found throughout the statement. Healthcare providers should ensure that their patients can and will use these devices correctly. This requires that the clinician: is aware of the devices that are currently available to deliver the prescribed drugs; knows the various techniques that are appropriate for each device; is able to evaluate the patient's inhalation technique to be sure they are using the devices properly; and ensures that the inhalation method is appropriate for each patient.

Experimental and theoretical screening of nanoscale oxide reactivity with LiBH4.

Experimentation, thermodynamic modeling, and atomic modeling were combined to screen the reactivity of SiO2, Al2O3, and ZrO2 nanoscale oxides with LiBH4. Equilibrium thermodynamic modeling showed that the reactions of oxides with LiBH4 could lead to formation of stable Li-bearing oxide and metal boride phases. Experimentation was conducted to evaluate the discharge/recharge reaction products of nanoscale oxide-LiBH4 mixtures. Thermal gravimetric analyses-mass spectroscopy and x-ray diffraction revealed significant SiO2 destabilization of LiBH4 dehydrogenation, resulting in the formation of lithium silicate and boric acid. A smaller amount of lithium metaborate and boric acid was formed with Al2O3. No destabilization products were observed with ZrO2. Density functional theory atomic modeling predicted much stronger LiBH4 interfacial adsorption on the SiO2 and Al2O3 surfaces than on the ZrO2 surface, which was consistent with the experimental findings. Following dehydrogenation, interfacial Li atoms were predicted to strongly adsorb on the oxide surfaces effectively competing with LiH formation. The interfacial Li interactions with Al2O3 and ZrO2 were equal in strength in the fully hydrided and dehydrided states, so that their predicted net effect on LiBH4 dehydrogenation was insignificant. Zirconia was selected for nanoframework development based on the combined observations of compatibility and weaker associative interactions with LiBH4.

Molecular interactions of the type 1 human immunodeficiency virus transregulatory protein Tat with N-methyl-d-aspartate receptor subunits.

We investigated the effect of type 1 human immunodeficiency virus (HIV-1) regulatory protein Tat on N-methyl-d-aspartate (NMDA) receptors expressed in Xenopus oocytes by voltage-clamp recording and its role in NMDA-mediated neurotoxicity using cultured rat hippocampal neurons. Tat (0.01-1muM) potentiated NMDA-induced currents of recombinant NMDA receptors. However, in the presence of Zn(2+), the potentiating effect of Tat was much more pronounced, indicating an additional Zn(2+)-related effect on NMDA receptors. Consistently, Tat potentiated currents of the particularly Zn(2+)-sensitive NR1/NR2A NMDA receptor with a higher efficacy, whereas currents from a Zn(2+)-insensitive mutant were only marginally augmented. In addition, chemical-modified Tat, deficient for metal binding, did not reverse Zn(2+)-mediated inhibition of NMDA responses, demonstrating that Tat disinhibits NMDA receptors from Zn(2+)-mediated antagonism by complexing the cation. We therefore investigated the interplay of Tat and Zn(2+) in NMDA-mediated neurotoxicity using cultures of rat hippocampal neurons. Zn(2+) exhibited a prominent rescuing effect when added together with the excitotoxicant NMDA, which could be reverted by the Zn(2+)-chelator tricine. Similar to tricine, Tat enhanced NMDA-mediated neurotoxicity in the presence of neuroprotective Zn(2+) concentrations. Double-staining with antibodies against Tat and the NR1 subunit of the NMDA receptor revealed partial colocalization of the immunoreactivities in membrane patches of hippocampal neurons, supporting the idea of a direct interplay between Tat and glutamatergic transmission. We therefore propose that release of Zn(2+)-mediated inhibition of NMDA receptors by HIV-1 Tat contributes to the neurotoxic effect of glutamate and may participate in the pathogenesis of AIDS-associated dementia.

Early pulmonary response to allergen is attenuated during acute emotional stress in females with asthma.

Some asthma patients and physicians who treat asthma have reported that stress worsens their disease. It has also recently been shown that chronic stressful life events increase airway inflammation 6-24 h after inhalation of antigen in patients with allergic asthma. However, there is no data regarding the effect of an acute stressor on the airway constriction that occurs within minutes of antigen inhalation (early pulmonary response) in this same population. The aim of this study was to examine this effect in eight females with allergic asthma. Each subject was challenged with increasing concentrations of inhaled allergen on a control visit (no stress) and on a stress visit, when they were asked to verbally recount an emotionally stressful situation between each concentration. Systolic (SP) and diastolic (DP) blood pressure, cardiac frequency (fC) and forced expiratory volume in one second (FEV1) were measured on both visits. SP, DP and fC were statistically increased on the stress visit as compared to control. Per cent decrease in FEV1 at the same last dose of allergen was significantly less on the stress visit (11.2 +/- 7.0%) compared to control (15.0 +/- 8.7%). These findings suggest that the early pulmonary response to inhaled allergen is attenuated while verbally re-experiencing an acute emotional stressor in females with allergic asthma.

Affinity labeling of cysteine-mutants evidences contact residues in modeled receptor binding sites.

To investigate the topology of binding sites in two ionotropic receptors, we have initiated a strategy combining affinity labeling with cysteine-scanning mutagenesis. For the GABAA receptor we have used reactive derivatives of non-competitive blockers (NCBs) to explore interacting positions in its channel. The polypeptide positions of the M2 segment of the alpha1 subunit which we mutated into cysteine were selected for their established accessibility, as determined by the substituted-cysteine accessibility method (SCAM). Using the Xenopus oocyte expression system, we show that receptors containing mutations V257C and S272C are inactivated by several reactive NCBs. These position-selective inactivations lead to an analysis of NCB binding in the channel. For the NMDA receptor glycine-binding site, the prototype antagonist L-701,324 was derivatized at different positions with different reactive groups. The receptor positions to mutate into cysteine were selected after a 3-D homology model. The observed receptor inactivations are mutant- and probe-selective, leading to an unambiguous chemical docking of the antagonist pharmacophore and supporting the model. The site-specificity of the inactivating reactions is assessed by protection experiments and by mutant to wild-type (WT) comparisons. The scope and limitations of the method are briefly discussed.

A pilot study examining the relationship between stress and serum cortisol concentrations in women with asthma.

The mechanism (s) by which stress exacerbates asthma is unknown. One explanation could be a reduction in endogenous serum cortisol concentrations as a result of stress. Our objective was to determine if a reduction in morning serum cortisol concentrations is associated with higher levels of stress in women with asthma. In this pilot study, seven women with a history of allergic-asthma were prospectively assigned to either low, moderate, or high stress groups based on a combination of their level of current stress and their resources to cope with the stress. After stress group assignment, women donated a morning blood sample, which was analyzed for serum cortisol concentration by an independent laboratory whose personnel were blinded to the subjects' stress status. Three women were assigned to the low stress group, two to the moderate stress group and two to the high stress group. Serum cortisol concentrations ranged from 8 to 23 microg/dl, averaging 14 +/- 6 microg/dl. A Spearman rank correlation indicated that serum cortisol concentrations were significantly inversely related to the stress groupings (r(s) = -0.915; P = 0.025). These results suggest that a reduction in morning serum cortisol concentration may be associated with higher levels of stress and lower resources to cope with the stress in women with allergic-asthma.

Mouthpiece diameter affects deposition efficiency in cast models of the human oral airways.

We examined the effect of altering mouthpiece diameter to 1.5, 2.0, and 2.7 cm on the deposition efficiency of inertial size particles (2, 4, and 8 microm) in adult human oral-pharyngeal-laryngeal (OPL) airway cast models at various inspiratory flow rates (30, 60, 90, and 120 L/min). Deposition efficiency of 2-microm particles was unaffected by changes in mouthpiece diameter at all flow rates. Deposition of 4-microm particles decreased significantly with the 2.0- and 2.7-cm mouthpieces compared to the 1.5 cm mouthpiece at 60, 90, and 120 L/min (p < 0.01). Deposition of 4-microm particles was significantly reduced with the 2.7-cm mouthpiece compared to the 2.0-cm mouthpiece at 90 and 120 L/min (p < 0.05). Deposition efficiency of 8 microm particles decreased significantly with the 2.0- and 2.7-cm mouthpieces compared to the 1.5-cm mouthpiece at 60 L/min (p < 0.05), and with the 2.7-cm mouthpiece compared to the 1.5-cm mouthpiece at 120 L/min (p < 0.05). These results suggest that the effect of mouthpiece diameter varies with particle size, with 2- and 8-microm particles least affected. However, our findings may have important implications for improving the future design of mouthpieces of devices that deliver particles with 4-microm diameters and require inspiratory flow rates of > or = 60 L/min (i.e., DPIs) for adequate drug delivery.

The organization of metabolic reaction networks. III. Application for diauxic growth on glucose and lactose.

A mathematical model to describe carbon catabolite repression in Escherichia coli is developed and in part validated. The model is aggregated from two functional units describing glucose and lactose transport and degradation. Both units are members of the crp modulon and are under control of a global signal transduction system which calculates the signals that turn on or off gene expression for the specific enzymes. Using isogenic mutant strains, our model is validated by a set of experiments. In these experiments, substrate composition of the preculture and of the experimental culture are varied in order to stimulate the system in different ways. With the obtained measurements (three states in the liquid phase and one intracellular component) a part of the model parameters could be estimated. Therefore all experiments could be sufficiently described with a single set of parameters.

Point mutations identify the glutamate binding pocket of the N-methyl-D-aspartate receptor as major site of conantokin-G inhibition.

Conantokin-G (Con-G), a gamma-carboxylglutamate (Gla) containing peptide derived from the venom of the marine cone snail Conus geographus, acts as a selective and potent inhibitor of N-methyl-D-aspartate (NMDA) receptors. Here, the effect of Con-G on recombinant NMDA receptors carrying point mutations within the glycine and glutamate binding pockets of the NR1 and NR2B subunits was studied using whole-cell voltage-clamp recording from cRNA injected Xenopus oocytes. At wild-type receptors, glutamate-induced currents were inhibited by Con-G in a dose-dependent manner at concentrations of 0.1-100 microM. Substitution of selected residues within the NR2B subunit reduced the inhibitory potency of Con-G, whereas similar mutations in the NR1 subunit had little effect. These results indicate a selective interaction of Con-G with the glutamate binding pocket of the NMDA receptor. Homology-based molecular modeling of the glutamate binding region based on the known structure of the glutamate binding site of the AMPA receptor protein GluR2 suggests how selected amino acid side chains of NR2B might interact with specific residues of Con-G.

Ubiquitination precedes internalization and proteolytic cleavage of plasma membrane-bound glycine receptors.

The inhibitory glycine receptor (GlyR) in developing spinal neurones is internalized efficiently upon antagonist inhibition. Here we used surface labeling combined with affinity purification to show that homopentameric alpha1 GlyRs generated in Xenopus oocytes are proteolytically nicked into fragments of 35 and 13 kDa upon prolonged incubation. Nicked GlyRs do not exist at the cell surface, indicating that proteolysis occurs exclusively in the endocytotic pathway. Consistent with this interpretation, elevation of the lysosomal pH, but not the proteasome inhibitor lactacystin, prevents GlyR cleavage. Prior to internalization, alpha1 GlyRs are conjugated extensively with ubiquitin in the plasma membrane. Our results are consistent with ubiquitination regulating the endocytosis and subsequent proteolysis of GlyRs residing in the plasma membrane. Ubiquitin-conjugating enzymes thus may have a crucial role in synaptic plasticity by determining postsynaptic receptor numbers.

Treating diabetes with aerosolized insulin.

Because of the pain, inconvenience, and disruption of lifestyle associated with the injection of insulin, many patients with diabetes are noncompliant in terms of treatment regimens that require daily multiple injections. To eliminate the pain and to improve treatment outcome, there has been increasing interest in the development of aerosolized insulin to replace subcutaneously (SC) delivered formulations. Recent studies in human volunteers have shown that when aerosolized insulin is effectively delivered to the alveolar region of the lung, absorption rates and decreases in glucose levels are similar to those achieved with SC-delivered insulin during the fasting state. Other human trials have shown that inhaled insulin also effectively controls postprandial glucose levels. Aerosolized insulin is well-tolerated, and there is no evidence of irritation, hypoglycemia, or changes in pulmonary function when administered over short periods. At present, limitations in the delivery device result in less efficient administration of insulin aerosol compared to SC dosing. However, new devices and different formulations of insulin, which are currently under development, should improve the efficiency. It is likely that the treatment of diabetes with aerosolized insulin will provide an effective alternative means for controlling plasma glucose levels in diabetic individuals. Aerosolized insulin also will serve as a developmental model for this route of administration for a number of other therapeutic peptides that are currently administered by injection only.

Gene therapy in cystic fibrosis.

Theoretically, cystic fibrosis transmembrane conductance regulator (CFTR) gene replacement during the neonatal period can decrease morbidity and mortality from cystic fibrosis (CF). In vivo gene transfers have been accomplished in CF patients. Choice of vector, mode of delivery to airways, translocation of genetic information, and sufficient expression level of the normalized CFTR gene are issues that currently are being addressed in the field. The advantages and limitations of viral vectors are a function of the parent virus. Viral vectors used in this setting include adenovirus (Ad) and adeno-associated virus (AAV). Initial studies with Ad vectors resulted in a vector that was efficient for gene transfer with dose-limiting inflammatory effects due to the large amount of viral protein delivered. The next generation of Ad vectors, with more viral coding sequence deletions, has a longer duration of activity and elicits a lesser degree of cell-mediated immunity in mice. A more recent generation of Ad vectors has no viral genes remaining. Despite these changes, the problem of humoral immunity remains with Ad vectors. A variety of strategies such as vector systems requiring single, or widely spaced, administrations, pharmacologic immunosuppression at administration, creation of a stealth vector, modification of immunogenic epitopes, or tolerance induction are being considered to circumvent humoral immunity. AAV vectors have been studied in animal and human models. They do not appear to induce inflammatory changes over a wide range of doses. The level of CFTR messenger RNA expression is difficult to ascertain with AAV vectors since the small size of the vector relative to the CFTR gene leaves no space for vector-specific sequences on which to base assays to distinguish endogenous from vector-expressed messenger RNA. In general, AAV vectors appear to be safe and have superior duration profiles. Cationic liposomes are lipid-DNA complexes. These vectors generally have been less efficient than viral vectors but do not stimulate inflammatory and immunologic responses. Another challenge to the development of clinically feasible gene therapy is delivery mode. Early pulmonary delivery systems relied on the direct instillation of aerosolized vectors, which can result in the induction of adverse reactions because vector is delivered into the lung parenchyma. More recent studies have examined the potential for using spray technologies to target aerosolized AAV vectors to the larger central airways, thereby avoiding alveolar exposure and adverse effects. Comparisons of lung deposition with nebulized delivery of aerosol and spray delivery indicate that spraying results in a more localized deposition pattern (predominantly in the proximal airways) and significantly higher deposition fractions than nebulization. These findings could lead to more efficient and targeted lung delivery of aerosolized gene vectors in the future.

Subunit-specific modulation of glycine receptors by neurosteroids.

The effects of pregnene and androstane steroids were studied on recombinant human glycine receptors (GlyRs) by whole-cell voltage-clamp electrophysiology. The 3beta-sulphates of pregnenolone (PREGS) and dehydroepiandrosterone (DHEAS) inhibited GlyR currents with K(I) values of 2-20 microM for different (alpha(1), alpha(2), alpha(4) and beta) GlyR subunits. PREGS resulted in a parallel shift of the response curve of glycine for alpha(1) GlyRs. The inhibitory potencies of DHEAS relative to PREGS were decreased in transition from embryonic alpha(2) towards adult alpha(1)beta GlyRs. A decreased potency of DHEAS for alpha(4) versus alpha(2) GlyRs represents the first pharmacological difference reported between these subunits. A negative charge at C3 is required for GlyR antagonism but androsterone sulphate epimers at C3 inhibited without stereoselectivity. Some point mutations of alpha(1) GlyRs with characteristic functional consequences did not significantly affect the inhibitory potency of PREGS. Progesterone selectively inhibited alpha(2) GlyRs, while PREG and its acetic ester potentiated alpha(1) GlyRs. Coexpression of the alpha subunits with the beta subunit eliminated the enhancing effects of PREG and attenuated the inhibitory potencies of the neurosteroids. Based on these data we propose that neurosteroids might modulate perinatal GlyR activity and thereby influence neuronal development.

Establishment of a novel in vitro system for studying the interaction of xenobiotic metabolism of liver and intestinal microflora.

We developed a new two-chamber system for the coculture of hepatocytes and fecal microflora under aerobic and anaerobic conditions, respectively, to investigate the sequential metabolism of chemicals by the liver and microflora in vitro. The culture device consisted of two chambers separated by a permeable polycarbonate membrane. In the aerobic compartment, hepatocytes were cultivated as a monolayer on the membrane and in the anaerobic compartment fecal microflora as a suspension. To characterize the metabolic capacity of the microflora and hepatocytes, various marker enzymes were studied. Azoreductase, nitroductase, beta-glucuronidase, beta-glucosidase and sulphatase were tested in the microflora of the feces from three volunteers who had had significantly different eating habits for years (daily meat, mixed diet, vegetarian). The microflora exhibited significant activities and the various enzymes differed only moderately in the samples from the three volunteers. For rat hepatocytes the activities of various cytochrome P450 forms and conjugating enzymes served as markers. The enzyme activities were tested in the coculture system during a 4-h culture period intended for the test protocol. Deethylation of ethoxycoumarin and 2alpha-, 6beta- and 16alpha-hydroxylation of testosterone decreased by about 30%, 25%, 40% and 20%, respectively, while there was no loss of glucuronidation and sulphonation of 3-OH-benzo(a)pyrene nor of glutathione conjugation of 1-chloro-2,4-dinitrobenzene during the 4-h culture period. The activities of the tested hepatic phase I and II enzymes were not changed after coculture of the hepatocytes with the microflora for 4 h. The applicability of the in vitro system for studying the metabolic interaction of liver and microflora was demonstrated using 7-ethoxycoumarin and the developmental drug EMD 57033, a thiadiazinon derivative from Merck KGaA, as model compounds. Both compounds were oxidized and conjugated by liver cells. In the coculture of hepatocytes and fecal microflora the resulting glucuronides and sulphoconjugates were split by hydrolytic enzymes of the intestinal microflora.

Targeting aerosol deposition in patients with cystic fibrosis: effects of alterations in particle size and inspiratory flow rate.

STUDY OBJECTIVE: To determine if aerosolized medications can be targeted to deposit in the smaller, peripheral airways or the larger, central airways of adult cystic fibrosis (CF) patients by varying particle size and inspiratory flow rate. DESIGN: Randomized clinical trial. SETTING: Outpatient research laboratory. PATIENTS: Nine adult patients with CF. INTERVENTIONS: Patients inhaled an aerosol comprised of 3.68+/-0.04 microm saline solution droplets (two visits) or 1.01+/- 0.2 microm saline solution droplets (two visits) for 30 s, starting from functional residual capacity and breathing at a slow or faster inspiratory flow rate. On all visits, the saline solution was admixed with the radioisotope (99m)Tc. Immediately after inhalation, a gamma camera recorded the deposition pattern of the radioaerosol in the lungs. Deposition images were analyzed in terms of the inner:outer zone (I:O) ratio, a measure of deposition in an inner zone (large, central airways) vs. an outer zone (small airways and alveoli). MEASUREMENTS AND RESULTS: For the 3.68-microm aerosol, I:O ratios averaged 2.29+/-1.45 and 2.54+/-1.48 (p>0.05), indicating that aerosol distribution within the lungs was unchanged while breathing at 12+/-2 L/min vs. 31+/-5 L/min, respectively. For the 1.01-microm aerosol, I:O ratios averaged 2.09+/-0.96 and 3.19+/-1.95 (p<0.05), indicating that deposition was predominantly in the smaller airways while breathing at 18+/-5 L/min and in the larger airways while breathing at 38+/-8 L/min, respectively. CONCLUSIONS: These results suggest that the targeted delivery of an aerosol to the smaller, peripheral airways or the larger, central airways of adult CF patients may be achieved by generating an aerosol comprised of approximately 1.0-microm particles and inspiring from functional residual capacity at approximately 18 L/min and approximately 38 L/min, respectively.

The relationship between particle deposition in the anterior nasal passage and nasal passage characteristics.

The objective of this study was to examine the effects of nasal passage characteristics on anterior particle deposition during cyclical breathing. Forty healthy, nonsmoking, adult subjects participated in this study. Nasal passage characteristics such as nostril length, width, angle, ellipticity, and minimum nasal cross-sectional area were measured. The subjects inhaled a polydisperse radioactively tagged aerosol (mass median aerodynamic diameter = 5.4 microns, geometric standard deviation [GSD] = 1.3) into the nose and exhaled through the mouth. The amount of radioactivity in the nose was measured immediately after inhalation and thereafter for 54 minutes. At 52.5 minutes, subjects wiped the accessible portion of the anterior nose to remove any remaining activity. The difference in activity at 52 and 54 minutes was used as a measure of activity removed during the nose wipe. Percentage of activity in the nasal passage at 52 minutes and percentage of activity removed with the nose wipe were considered surrogates for particles deposited in the anterior nasal passage. A multiple regression analysis showed that the degree of ellipticity of the nostrils was significantly related to particle deposition in the anterior nasal passage. These results suggest that ellipticity of the nostrils may be a determinant of the amount of particle deposition in the anterior nasal passage.

Potent bronchoprotective effect of deep inspiration and its absence in asthma.

In the absence of deep inspirations, healthy individuals develop bronchoconstriction with methacholine inhalation. One hypothesis is that deep inspiration results in bronchodilation. In this study, we tested an alternative hypothesis, that deep inspiration acts as a bronchoprotector. Single-dose methacholine bronchoprovocations were performed after 20 min of deep breath inhibition, in nine healthy subjects and in eight asthmatics, to establish the dose that reduces forced expiratory volume in 1 s by >15%. The provocation was repeated with two and five deep inspirations preceding methacholine. Additional studies were carried out to assess optimization and reproducibility of the protocol and to rule out the possibility that bronchoprotection may result from changes in airway geometry or from differential spasmogen deposition. In healthy subjects, five deep inspirations conferred 85% bronchoprotection. The bronchoprotective effect was reproducible and was not attributable to increased airway caliber or to differential deposition of methacholine. Deep inspirations did not protect the bronchi of asthmatics. We demonstrated that bronchoprotection is a potent physiologic function of lung inflation and established its absence, even in mild asthma. This observation deepens our understanding of airway dysfunction in asthma.

Glycine receptors containing the alpha4 subunit in the embryonic sympathetic nervous system, spinal cord and male genital ridge.

Inhibitory glycine receptors (GlyRs) are known to mediate postsynaptic inhibition in spinal cord, brain stem and some higher brain regions. Several developmentally and regionally regulated GlyR isoforms exist, which result from a differential expression of the GlyR alpha (alpha1-alpha4) and beta subunit genes. Currently, very little is known about GlyRs containing the alpha4 subunit, whose existence was predicted from a partial genomic sequence. Here, we describe the isolation of complementary DNA (cDNA) sequences for the mouse and chick GlyR alpha4 subunits. We show that a mouse GlyR alpha4 subunit full-length cDNA directs the formation of functional homo-oligomeric strychnine-sensitive GlyRs in Xenopus laevis oocytes and mammalian cells, and that these resemble GlyRs composed of the alpha1 subunit in pharmacological profile and single-channel properties. In situ hybridization reveals high levels of GlyR alpha4 subunit transcripts in the embryonic (E13) chick spinal cord, lumbosacral sympathetic ganglia and dorsal root ganglia. The avian GlyR alpha4 subunit gene also shows male-specific expression in the developing genital ridge. The pharmacological profile of alpha4 subunit-containing receptors and deduced location of the avian GlyR alpha4 subunit are consistent with it being a component of the embryonic excitatory GlyRs previously identified in sympathetic neurons. Our data also suggest a novel role for GlyRs in the maturation of reproductive organs.

Subunit-dependent inhibition of recombinant rodent N-methyl-D-aspartate receptors by a HIV-1 glycoprotein 120 derived peptide.

Considerable evidence suggests that low (picomolar) concentrations of the HIV-1 envelope glycoprotein gp120 induce neuronal cell death by stimulating the release of microglial toxins, which in turn activate N-methyl-D-aspartate (NMDA) receptors. Conversely, high (micromolar) concentrations of gp120 have been reported to directly inhibit NMDA receptor-mediated currents and do not induce neurotoxicity. Here we show that micromolar concentrations of a synthetic peptide corresponding to the V3-loop of gp120 (V3-pep) inhibited agonist responses of recombinant heteromeric rodent NMDA receptors expressed in Xenopus laevis oocytes by decreasing their apparent glycine affinity. Different combinations of NMDA receptor subunits displayed differential sensitivities to inhibition by V3-pep, with a potency rank order of NR1/2B > NR1/2D > NR1/2C > or = NR1/2A. Our observations may provide an explanation for the reduced neurotoxicity of high doses of gp120 in cell cultures and may be useful for the pharmacological discrimination of NMDA receptor subtypes.

Kinetic and mutational analysis of Zn2+ modulation of recombinant human inhibitory glycine receptors.

1. The effects of Zn2+ on glycine receptor (GlyR) currents were analysed in Xenopus oocytes and human embryonic kidney cells expressing homomeric human wild-type and mutant alpha1 subunit GlyRs. 2. Low concentrations (10 microM) of extracellular Zn2+ converted the partial agonist taurine into a high-efficacy agonist. Concentration-response analysis showed that the EC50 for taurine decreased whereas the Hill coefficient increased under these conditions. In contrast, 50-500 microM Zn2+ showed an increased EC50 value and reduced maximal inducible taurine currents. The potency of competitive antagonists was not affected in the presence of Zn2+. 3. Single-channel recording from outside-out patches revealed different kinetics of glycine- and taurine-gated currents. With both agonists, Zn2+ altered the open probability of the alpha1 GlyR without changing its unitary conductance. Low Zn2+ concentrations (5 microM) increased both the opening frequency and mean burst duration, whereas higher Zn2+ concentrations (> 50 microM) reduced GlyR open probability mainly by decreasing the open frequency and the relative contribution of the longest burst of the single-channel events. 4. Site-directed mutagenesis of the GlyR alpha1 subunit identified aspartate 80 and threonine 112 as important determinants of Zn2+ potentiation and inhibition, respectively, without affecting potentiation by ethanol. 5. Our data support the view that Zn2+ modulates different steps of the receptor binding and gating cycle via specific allosteric high- and low-affinity binding sites in the extracellular N-terminal region of the GlyR alpha1 subunit.