Selective alterations in binding kinetic parameters and allosteric regulation of N-methyl-D-aspartate receptors after prolonged seizures in the developing rat brain

To investigate the effect of chronic smoke exposure on pulmonary macrophages (PM), the expression of seven different surface and intracellular molecules of PM was studied in induced sputum (IS) samples from healthy volunteers--nine smokers and seven non-smokers. Sputum was induced by inhalation of nebulized saline (3.5% NaCl). Cell viability and total cell counts (TCC) were performed immediately. Cell differentials were determined on May-Grunwald Giemsa-stained cytospin preparations. The PM were immunologically characterized by use of the following monoclonal antibodies: RFD1, RFD7, CD11b, CD54, CD68, CD71 and HLA-DR. The stainings were performed with a three-step, indirect immuno-alkaline phosphate method. Viability and TCC did not differ between the groups. Smokers had a higher percentage of macrophages (P < 0.05) and a lower proportion of neutrophils (P < 0.05). The percentage of macrophages expressing RFD1, HLA-DR, CD71 (P < 0.01 for all) and CD54 (P < 0.05) was significantly lower in smokers, whereas the remaining markers were expressed equally in the two groups. The results indicate that smoking induces a decrease in the expression by PM of surface molecules known to be associated with the antigen-presenting function.     

Modulation of N-methyl-D-aspartate receptor function by glycine transport

The recent discovery of glycine transporters in both the central nervous system and the periphery suggests that glycine transport may be critical to N-methyl-D-aspartate receptor (NMDAR) function by controlling glycine concentration at the NMDAR modulatory glycine site. Data obtained from whole-cell patch-clamp recordings of hippocampal pyramidal neurons, in vitro, demonstrated that exogenous glycine and glycine transporter type 1 (GLYT1) antagonist selectively enhanced the amplitude of the NMDA component of a glutamatergic excitatory postsynaptic current. The effect was blocked by 2-amino-5-phosphonovaleric acid and 7-chloro-kynurenic acid but not by strychnine. Thus, the glycine-binding site was not saturated under the control conditions. Furthermore, GLYT1 antagonist enhanced NMDAR function during perfusion with medium containing 10 microM glycine, a concentration similar to that in the cerebrospinal fluid in vivo, thereby supporting the hypothesis that the GLYT1 maintains subsaturating concentration of glycine at synaptically activated NMDAR. The enhancement of NMDAR function by specific GLYT1 antagonism may be a feasible target for therapeutic agents directed toward diseases related to hypofunction of NMDAR.     

Modulation of intrinsic circuits by serotonin 5-HT3 receptors in developing ferret visual cortex

Serotonergic projections are widespread in the developing neocortex, but their functions are obscure. The effects of 5-HT3 receptor agonists on cortical circuit response properties were studied in slices of ferret primary visual cortex using high-speed optical imaging of voltage-sensitive dye signals and whole-cell patch-clamp recording. Activation of the 5-HT3 receptor decreased the amplitude and lateral extent of excitation throughout postnatal development. This effect peaks after eye opening, which indicates a function for serotonergic modulation of circuit responses during the period of refinement of cortical connections. Whole-cell patch-clamp recordings from single neurons revealed that synaptic responses evoked by white matter stimulation were reduced by 5-HT3 receptor agonists, whereas the frequency of spontaneous GABAergic synaptic currents was enhanced dramatically. This indicates that the modulation of spontaneous synaptic activity by fast-acting serotonin receptors is reflected in an inhibition of the circuit response, in line with the notion of background synaptic activity altering the spatiotemporal integration properties of cortical cells by changing their membrane potential and their electrotonic structure. These mechanisms may regulate the response properties of intrinsic circuits in both the adult and developing neocortex.     

Androgen and estrogen receptors in the developing mouse brain

Specific binding of the androgens, 5alpha-dihydrotestosterone (DHT) and testosterone, and of 17beta-estradiol by brain cytosol from mice at 3-5,9-11, and 18-23 days of age was measured by charcoal assay and glycerol gradient centrifugation and analyzed by Scatchard plots. The immature mouse brain contains putative receptors for these steroids which migrate at 8 S in gradients at low ionic strength and at 5 S in 0.5 M KCl. Investigation of estradiol binding was complicated by the presence in cytosol from 3-5 day-old mice, and to a lesser extent from 9-11 day-old mice, of the high capacity, fetoneonatal estradiol binding protein (FEBP) which is no longer detectable at 3 weeks. The rapid dissociation of the FEBP-estradiol complex under non-equilibrium conditions probably led to over-estimation of free steroid concentration and thus to an apparent increase in the affinity of 8 S receptor for estradiol with age (for female brain cytosol KD=9.5 X 10(-10)M at 3-5 days and 2.7 X 10(-10)M at 18-23 days). The number of estradiol binding sites remains relatively constant during the first 3 weeks at 7-9 fmol/mg protein, while the number of DHT binding sites in female brain increases from 3.2+/-0.3 to 6.6+/-0.9 to 9.6+/-0.3 (mean+/-SE) fmol/mg protein in the 3 age groups. Dissociation constants and numbers of sites for both DHT and estradiol binding are similar in brain cytosol from male and female mice. Testosterone and DHT compete for the same binding site, but its affinity for DHT is about twice that for testosterone. The high affinity of the brain receptor for DHT (KD=4-5 X 10(-10)M) may reflect the slow metabolism of DHT to 5alpha-androstanediols, amounting to less than 10% after 2 h at 0 C. Binding of DHT and estradiol to cytosol from brain regions was also investigated. DHT receptors increase in parallel in various regions with age; the concentration of sites in the hypothalamus-preoptic area (HPOA) is 1.2-3.4 times that in the cerebral cortex (C). The concentration of estradiol binding sites in HPOA to that in C increases about 12-fold from neonatal to adult stages, reflecting both an increase in HPOA sites and a decrease in C sites, while the concentration in the remainder of the brain shows little change. Androgen and estrogen receptors in brain cytosol from immature mice can be distinguished by their different specificities and developmental patterns in whole brain and brain regions. The presence and properties of these receptors in the brain of neonatal mice are discussed with respect to their possible role in sexual differentiation of the brain.     

Evidence for functional co-activation of N-methyl-D-aspartate receptors by glycine

The excitatory neurotransmitter glutamate acts at several receptor subtypes in the CNS, including N-methyl-D-aspartate (NMDA) receptors. The inhibitory neurotransmitter glycine is a co-agonist of NMDA receptors and functional glycinergic co-activation of NMDA receptors is theoretically possible due to the presence of background glutamate. We report the first high fidelity recording of a distinct NMDA receptor excitatory post-synaptic potential (EPSP) in the intact mammalian CNS. The EPSP was evoked by multi-shock activation of a glycinergic pathway rather than a glutamatergic pathway and had characteristics most compatible with glycine, rather than glutamate, activation of NMDA receptors.     

Blockade of NMDA receptors and apoptotic neurodegeneration in the developing brain

Programmed cell death (apoptosis) occurs during normal development of the central nervous system. However, the mechanisms that determine which neurons will succumb to apoptosis are poorly understood. Blockade of N-methyl-D-aspartate (NMDA) glutamate receptors for only a few hours during late fetal or early neonatal life triggered widespread apoptotic neurodegeneration in the developing rat brain, suggesting that the excitatory neurotransmitter glutamate, acting at NMDA receptors, controls neuronal survival. These findings may have relevance to human neurodevelopmental disorders involving prenatal (drug-abusing mothers) or postnatal (pediatric anesthesia) exposure to drugs that block NMDA receptors.     

Modulation of the N-methyl-D-aspartate receptor by haloperidol: NR2B-specific interactions

The dopaminergic antagonist haloperidol has an eight- to 10-fold higher affinity for NMDA receptors containing the NR2B (epsilon2) subunit, showing the same subunit specificity as ifenprodil, polyamines, and magnesium. In the present study, we have compared the effects of mutations altering polyamine and ifenprodil sensitivity on haloperidol sensitivity of NMDA receptors. As seen for spermidine stimulation, high-affinity haloperidol inhibition is governed by the region around amino acid 198, based on results from chimeric murine NR2A/NR2B (epislon1/epsilon2) receptors. Mutation of epsilon2E201 in this region to asparagine or arginine causes a 10-fold decrease in the ability of haloperidol to inhibit 125I-MK-801 binding. Epsilon2E201 does not govern the interactions of ifenprodil, because all of the mutants at epsilon2E201 exhibited wild-type affinity for ifenprodil. Mutation of epsilon2R337 causes a 400-fold loss in apparent affinity for ifenprodil but does not change the effects of haloperidol. The structural determinants of spermidine stimulation do not perfectly match those for haloperidol inhibition, as mutations of E200 remove haloperidol inhibition but do not alter polyamine stimulation. The present results thus demonstrate that although spermidine, haloperidol, and ifenprodil share subunit selectivity and overlapping pharmacology, they also have specific structural determinants.     

Modulation of neuronal migration by NMDA receptors

The N-methyl-D-aspartate (NMDA) subtype of the glutamate receptor is essential for neuronal differentiation and establishment or elimination of synapses in a developing brain. The activity of the NMDA receptor has now been shown to also regulate the migration of granule cells in slice preparations of the developing mouse cerebellum. First, blockade of NMDA receptors by specific antagonists resulted in the curtailment of cell migration. Second, enhancement of NMDA receptor activity by the removal of magnesium or by the application of glycine increased the rate of cell movement. Third, increase of endogenous extracellular glutamate by inhibition of its uptake accelerated the rate of cell migration. These results suggest that NMDA receptors may play an early role in the regulation of calcium-dependent cell migration before neurons reach their targets and form synaptic contacts.     

Calmodulin mediates calcium-dependent inactivation of N-methyl-D-aspartate receptors

Ca2+ influx through N-methyl-D-aspartate (NMDA) receptors activates signal transduction pathways critical for many forms of synaptic plasticity in the brain. NMDA receptor-mediated Ca2+ influx also downregulates the gating of NMDA channels through a process called Ca2+-dependent inactivation (CDI). Recent studies have demonstrated that the calcium binding protein calmodulin directly interacts with NMDA receptors, suggesting that calmodulin may play a role in CDI. We report here that the mutation of a specific calmodulin binding site in the CO region of the NR1 subunit of the NMDA receptor blocks CDI. Moreover, intracellular infusion of a calmodulin inhibitory peptide markedly reduces CDI of both recombinant and neuronal NMDA receptors. Furthermore, this inactivating effect of calmodulin can be prevented by coexpressing a region of the cytoskeletal protein alpha-actinin2 known to interact with the CO region of NR1. Taken together, these results demonstrate that the binding of Ca2+/calmodulin to NR1 mediates CDI of the NMDA receptor and suggest that inactivation occurs via Ca2+/calmodulin-dependent release of the receptor complex from the neuronal cytoskeleton.     

Modulation of neuropeptide FF receptors by guanine nucleotides and cations in membranes of rat brain and spinal cord

Using a radioligand binding assay, we examined ionic modulation and G protein coupling of neuropeptide FF (NPFF) receptors in membranes of rat brain and spinal cord. We found that NaCl (but not KCl or LiCl) and MgCl2 increased specific 125I-YLFQPQRFamide (125I-Y8Fa) binding to NPFF receptors in both tissues in a dose-dependent manner, with optimal conditions being 60 mM NaCl and 1 mM MgCl2. Guanine nucleotides dose-dependently inhibited specific 125I-Y8Fa binding to rat brain and spinal cord membranes with maximal effects of 64 +/- 6 and 71 +/- 2%, respectively. The order of potency was nonhydrolyzable GTP analogues > GTP > or = GDP > GMP, ATP. The guanine nucleotide inhibition was observed in the absence and presence of NaCl and MgCl2. The mechanism of inhibition in spinal cord membranes appeared to be a reduction in the number of NPFF receptors; in one experiment, control KD and Bmax values were 0.068 nM and 7.2 fmol/mg of protein, respectively, and with 0.1 microM guanylylimidodiphosphate the respective values were 0.081 nM and 4.9 fmol/mg, a 32% reduction in receptor number. Similar results were obtained with guanosine 5'-O-(3-thiotriphosphate). Our data suggest that 125I-Y8Fa binding sites in rat CNS are G protein-coupled NPFF receptors regulated by GTP and cations.     

Prevention by magnesium of excitotoxic neuronal death in the developing brain: an animal model for clinical intervention studies

Excitotoxic disturbances during brain development were studied in the mouse using intracerebral injections of ibotenate, a glutamatergic agonist of the N-methyl-D-aspartate (NMDA) complex receptor, to analyse the protective effect of a systemic bolus of MgSO4, a non-competitive antagonist of the NMDA ionophore-complex receptor. MgSO4 did not prevent microgyia, induced by ibotenate when injected at P0 immediately after the post-migratory settlement of layer V, but did prevent ulegyrias, porencephalic cysts, and other cortical and cortical-subcortical hypoxic-like lesions arising after completion of the neocortical cyto-architectonic development at P5. Protection was optimal in 80 per cent of mice at 600mg/kg, with no mortality due to MgSO4; thereafter mortality increased with dosage. The protective effect appears after the developmental acquisition of two properties of the excitotoxic cascade, namely the coupling of the massive calcium influx with NMDA-receptor overstimulation and the predominance of magnesium-obliterable calcium channels. This animal model supports the clinical intervention studies with magnesium in hypoxias/perfusion failures and has implications for their design. If maturation of the excitotoxic cascade follows the same sequence in humans, protection is probably low before 26 weeks of gestational age.     

Modulation of recombination human gamma-aminobutyric acidA receptors by isoflurane: influence of the delta subunit

BACKGROUND: The gamma-aminobutyric acid (GABA)A receptor/chloride channel has a broad-spectrum anesthetic sensitivity and is a key regulator of arousal. Each receptor/channel complex is an assembly of five protein subunits. Six subunit classes have been identified, each containing one to six members; many combinations are expressed throughout the brain. Benzodiazepines and intravenous anesthetic agents are clearly subunit dependent, but the literature to date suggests that volatile anesthetics are not. The physiological role of the delta subunit remains enigmatic, and it has not been examined as a determinant of anesthetic sensitivity. METHODS: Combinations of GABA(A) receptor subunit cDNAs were injected into Xenopus laevis oocytes: alpha1beta1, alpha1beta1gamma2L, alpha1beta1delta, and alpha1beta1gamma2Ldelta. Expression of functional ion channels with distinct signalling and pharmacologic properties was demonstrated within 1-4 days by established electrophysiological methods. RESULTS: Co-expression of the delta subunit produced changes in receptor affinity; current density; and the modulatory efficacy of diazepam, zinc, and lanthanum; it also produced subtle changes in the rate of desensitization in response to GABA. Isoflurane enhanced GABA-induced responses from all combinations: alphabeta delta (>10-fold) > alphabeta > alphabeta gamma > or = alphabeta gammadelta (approximately 5-fold). Dose-response plots were bell shaped. Compared with alphabeta gamma receptors (EC50 = 225 microM), both alphabeta delta (EC50 = 372 microM) and alphabeta gammadelta (EC50 = 399 microM) had a reduced affinity for isoflurane. Isoflurane (at a concentration close to the EC50 for each subunit) increased the affinity of GABA for its receptor but depressed the maximal response (alphabeta gamma and alphabeta gammadelta). In contrast, the small currents through alphabeta delta receptors were enhanced, even at saturating agonist concentrations. CONCLUSIONS: Delta subunit expression alters GABA(A) receptor function but is not an absolute determinant of anesthetic sensitivity.     

Activation of 5-HT2 receptors facilitates depolarization of neocortical neurons by N-methyl-D-aspartate

We prospectively studied 63 children with transient hyperglycemia to determine their risk of acquiring insulin-dependent diabetes mellitus (IDDM) and to evaluate the predictive value of immunologic markers of prediabetes and of the intravenous glucose tolerance test. Children with transient hyperglycemia were identified by a prospective systematic review of the laboratory reports of a large children's hospital and an office-based pediatric practice and by referral from pediatricians. Transient hyperglycemia occurred in 0.46% of children seen in the children's hospital and in 0.013% of children attending a pediatric office practice. Insulin-dependent diabetes mellitus developed within 18 months of identification in 32% of children in whom transient hyperglycemia was discovered in the absence of a serious illness, compared with 2.3% of children identified during a serious illness (relative risk, 13.9; 95% confidence interval, 1.56 to 123.5). Islet cell antibodies and competitive insulin autoantibodies each had a 100% positive predictive value for IDDM; the negative predictive value of islet cell antibodies and competitive insulin autoantibodies was 96% and 98%, respectively. The stimulated insulin release during an intravenous glucose tolerance test, adjusted for age, had the highest overall accuracy of prediction. All children less than 6 years of age with stimulated insulin release levels < 85 pmol/L (12 microU/ml) subsequently had IDDM, as did an 11-year-old child whose stimulated insulin release level was below the 1st percentile of 170 pmol/L (24 microU/ml). To date, no child whose stimulated insulin release level was above the 5th percentile has had IDDM. We conclude that when transient hyperglycemia occurs during a serious intercurrent illness, the risk of progression to IDDM is low. In contrast, one third of children in whom transient hyperglycemia is identified without a serious illness can be expected to have IDDM within 1 year. A combination of islet cell antibodies, competitive insulin autoantibodies, and stimulated insulin release levels during an intravenous glucose tolerance test can accurately distinguish children with prediabetes from those with presumed benign transient increases in plasma glucose concentrations.     

Modulation of calcium by inhibitory systems in the developing auditory midbrain

Inhibitory synaptic transmission is of fundamental importance during the maturation of central auditory circuits, and their subsequent ability to process acoustic information. The present study investigated the manner in which inhibitory transmission regulates intracellular free calcium levels in the gerbil inferior colliculus using a brain slice preparation. Inhibitory and excitatory postsynaptic potentials were evoked by electrical stimulation of the ascending afferents at the level of the dorsal nucleus of the lateral lemniscus. Pharmacologically isolated inhibitory synaptic potentials were able to attenuate a calcium rise in collicular neurons that was generated by depolarizing current injection. In addition, GABA(A) and glycine receptor antagonists typically led to an increase of calcium in collicular neurons during electrical stimulation of the ascending afferent pathway at the level of the dorsal nucleus of the lateral lemniscus. Bath application of GABA or muscimol, a GABA(A) receptor agonist, evoked a brief hyperpolarization followed by a long-lasting depolarization in inferior colliculus neurons. This treatment also induced a transient calcium increase that correlated with the membrane depolarization phase. Baclofen, a GABA(B) receptor agonist, had no effect on either membrane potential or calcium levels. Ratiometric measures indicated that the muscimol-evoked rise in calcium was approximately 150 nM above basal levels. The muscimol-evoked responses were completely antagonized by bicuculline and attenuated by picrotoxin. Together, these results suggest that inhibitory synaptic transmission participates in the regulation of postsynaptic calcium during the developmental period. Inhibitory transmission may attenuate a calcium influx that is evoked by excitatory synapses, but it can also produce a modest influx of calcium when activated alone. These mechanisms may help to explain the influence of inhibitory transmission on the development of postsynaptic properties.     

Effect of spinal cord transection on N-methyl-D-aspartate receptors in the cord

Spinal cord injury can lead to an exaggeration of transmission through spinal pathways, resulting in muscle spasticity, chronic pain, and abnormal control of blood pressure and bladder function. These conditions are mediated, in part, by N-methyl-D-aspartate (NMDA) receptors on spinal neurons, but the effects of cord injury on the expression or function of these receptors is unknown. Therefore, antibodies to the NMDA-R1 receptor subunit and binding of [3H]MK-801 were used to assess NMDA receptors in the spinal cord. Receptor density in rats with intact spinal cords was compared to that in rats 1 and 2 weeks after spinal cord transection (SCT) at the mid-thoracic level. At 1 and 2 weeks after SCT, [3H]MK-801 binding was reduced in most laminae in cord segments caudal to the injury, whereas no decrease in amount of R1 subunit immunoreactivity was observed. No significant changes in [3H]MK-801 binding and NMDA-R1 immunoreactivity could be seen rostral to the transection. Since [3H]MK-801 binding requires an open ion channel, the discrepancy between [3H]MK-801 binding and immunocytochemistry may indicate a loss of functional receptors without a consistent change in their total number. Therefore, the exaggerated reflexes that are well established in rats 2 weeks after cord injury must be mediated by a mechanism that withstands attenuation of NMDA receptor function.     

Modulation of the discriminative stimulus and rate-altering effects of cocaine by competitive and noncompetitive N-methyl-D-aspartate antagonists

Preschool antecedents of the use of defense mechanisms were longitudinally studied using data from 90 nursery school children who were again evaluated at age 23. Defense use was determined by coding Thematic Apperception Test (H.A. Murray, 1943) stories with a method previously shown to be reliable and valid. The findings indicated continuity between preschool personality and subsequent defense use for male participants but little continuity for female participants. Young men's use of the age-inappropriate defense of denial was predicted by indications at ages 3-4 of low ego resiliency and psychological difficulties in the areas of emotion, intellect, impulse control, and social interactions.     

Muscarinic receptors in developing rat colon

Muscarinic receptor subtypes were characterized in fetal (21 day), newborn (3 day), and adult (3 month) rat colon smooth muscle. Saturation binding of the nonselective muscarinic antagonist radioligand [3H]quinuclidinyl benzilate revealed a single class of binding sites in all three age groups. The binding affinities of [3H]quinuclidinyl benzilate were not significantly different among three age groups (KD: 0.19-->0.27 nM). In contrast, the receptor densities (Bmax, fmol/mg protein) showed a significant age-related decrease with fetus (518.9 +/- 7.4) > newborn (480.3 +/- 45.6) > adult (192.4 +/- 32.8). In both newborn and adult tissues, the muscarinic agonist carbachol bound to two sites with high and low affinities. Although the agonist binding affinities in the newborn tissue were not significantly different from those in the adult tissue, the high-affinity binding sites for carbachol were significantly increased in the later (41%-->61%). Addition of guanosine-5'-O-(3-thio)triphosphate (100 microM) abolished apparent high-affinity binding sites in both newborn and adult tissues. Antagonist competition binding in the newborn tissue indicated a homogeneous population of muscarinic M2 receptors. Unlike in newborn tissues, the heterogeneous binding of pirenzepine and 4-diphenylacetoxy-N-methylpiperidine methobromide in adult tissues revealed coexistence of muscarinic M3 (45%) and M2 (55%) receptors. In accordance, activation of muscarinic receptors in the adult tissue stimulated synthesis of inositol 1,4,5-trisphosphate. These results suggest maturational changes of muscarinic receptor subtypes and their coupling to G proteins in rat colonic smooth muscle. These changes may account, at least in part, for developmental alterations of functional responses in colonic smooth muscle.     

Quantitation of benzodiazepine receptors in human brain using the partial saturation method

The possible relationship between different patterns of esterases and the resistance to various types of insecticides was analyzed by bioassays, biochemical tests, and electrophoresis carried out in a population of Culex quinquefasciatus from Camagüey and in three colonies obtained from it by family selection. The original population was heterogeneous and presented 8 combinations of esterases in polyacrylamide gel. A3A6B6, B1B6 and B1A6B6 were the most frequent of them and characterized each selected colony. Every studied colony, including the parental one, showed different levels of resistance to diverse insecticides. Only the resistance to propoxur showed less variation. In every colony it seems to exist a combination of resistance mechanisms; however, the differences found in tests with DEF synergized insecticides show that the different bands of zymograms may represent esterases contributing in a different way to resistance.     

Modulation of agonist and antagonist interactions in serotonin 1A receptors by alcohols

The serotonin type IA (5-HT1A) receptors are members of a superfamily of seven transmembrane domain receptors that couple to GTP binding regulatory proteins (G-proteins). Serotonergic signalling has been shown to play an important role in alcohol tolerance and dependence. We have studied the effects of alcohols on ligand (agonist and antagonist) binding to bovine hippocampal 5-HT1A receptor in native as well as solubilized membranes. Our results show that alcohols inhibit the specific binding of the agonist OH-DPAT and the antagonist p-MPPF to 5-HT1A receptors in a concentration-dependent manner.