The Effect of Barium on [3H]Noradrenalin Release from the Human Neuroblastoma SH-SY5Y

Replacement of Ca²⁺ with Ba²⁺ in HEPES-buffered saline stimulated [³H]noradrenalin release in the human neuroblastoma clone SH-SY5Y by up to 20% of the cell content in the absence of other secretory stimuli. The Ba²⁺-evoked release was inhibited by 85% by 3 μM tetrodotoxin and 95% by 5 μM nifedipine. Ba²⁺ also increased the potency of K⁺-evoked release of [³H]noradrenalin, as maximal release was observed with 60 mM K⁺ compared with the 100 mM K⁺ necessary to achieve maximal release in the presence of Ca²⁺. In contrast, replacing Ca²⁺ with Ba²⁺ had little effect on carbachol- and bradykinin-evoked release of [³H]noradrenalin. No evidence was obtained from studies on changes in [Ca²⁺]_i (in response to 100 pM carbachol) using fura-2 that Ba²⁺ could enter intracellular stores in SH-SY5Y cells. Whole-cell patch-clamp studies showed that Ba²⁺ depolarizes SH-SY5Y cells as well as enhancing inward Ca²⁺ channel currents and shifting their voltage dependence to more negative values. These results are discussed in terms of the hypothesis that Ba²⁺ blocks K⁺ channels, leading to depolarization followed by opening of voltage-sensitive Na⁺ channels. This in turn opens voltage-sensitive L-type Ca²⁺ channels, which are coupled to the release of [³H]noradrenalin in SH-SY5Y cells.     

HIV-1 Envelope Proteins gp120 and gp160 Potentiate NMDA-induced [Ca2+]i Increase, Alter [Ca2+]i Homeostasis and Induce Neurotoxicity in Human Embryonic Neurons

The envelope glycoprotein gp120 of the human immunodeficiency virus HIV-1 has been proposed to cause neuron death in developing murine hippocampal cultures and rat retinal ganglion cells. In the present study, cultured human embryonic cerebral and spinal neurons from 8- to 10-week-old embryos were used to study the neurotoxic effect of gp120 and gp160. Electrophysiological properties as well as N -methyl- d -aspartate (NMDA)-induced currents were recorded from neurons maintained in culture for 10–30 days. Neither voltage-activated sodium or calcium currents nor NMDA-induced currents were affected by exposure of neurons to 250 pM gp120 or gp160. In contrast, when neurons were subjected to photometric measurements using the calcium dye indo-1 to monitor the intracellular free Ca²⁺ concentration ([Ca²⁺]_i), gp120 and gp160 (20–250 pM) potentiated the large rises in [Ca²⁺]_i induced by 50 μM NMDA. The potentiation of NMDA-induced Ca²⁺ responses required the presence of Ca²⁺ in the medium, and was abolished by the NMDA antagonist d -2-amino-5-phosphonovalerate (AP5) and the voltage-gated Ca²⁺ channel inhibitor nifedipine. Moreover, exposure of a subpopulation of spinal neurons (25% of the cells tested) to 20–250 pM gp120 or gp160 resulted in an increase in [Ca²⁺]_i that followed three patterns: fluctuations not affected by AP5, a single peak, and the progressive and irreversible rise of [Ca²⁺]_i. The neurotoxicity of picomolar doses of gp120 and gp160 cultures was estimated by immuno-fluorescence and colorimetric assay. Treatment of cultures with AP5 or nifedipine reduced gp120-induced toxicity by 70 and 100% respectively.     

Release of Acetylcholine and Noradrenaline from the Cholinergic and Adrenergic Afferents in Rat Hippocampal CA1, CA3 and Dentate Gyrus Regions

An attempt was made to study the release of acetylcholine (ACh) and noradrenaline and their presynaptic modulation in isolated slice preparations dissected from different subfields of the hippocampus: CA1, CA3 and the dentate gyrus. The slices were perfused and loaded with [³H]choline or with [³H]noradrenaline. The release in response to field stimulation was determined radiochemically and the content of transmitters was assayed by a chemiluminescent method or by HPLC combined with electrochemical detection. After 30 min of loading with [³H]choline there were marked subregional differences in the specific activity of [³H]ACh content. The highest concentration was measured in the dentate gyrus and the lowest in CA3. Evidence was obtained that in all three subfields the cholinergic axon terminals are equipped with inhibitory muscarinic autoreceptors and the noradrenergic terminals with α₂-autoreceptors, as indicated by an increase in transmitter release when the tissue was exposed to selective muscarinic or α₂-adrenoceptor antagonists. In contrast, the cholinergic boutons are not equipped with α₂-adrenoceptors, and noradrenergic terminals do not possess inhibitory muscarinic receptors. It is therefore concluded that while the release of both ACh and noradrenaline is controlled by negative feedback modulation, there is no possibility of establishing a presynaptic inhibitory interaction between the two.     

Effects of P-Aminophenyl Dichloroarsine on Reduced High-affinity [3H]Nicotine Binding Sites from Chick Brain: A Covalent,Yet Reversible,Agent for Neuronal Nicotinic Receptors

Neuronal nicotinic acetylcholine receptor (nAChR) α-subunits contain a conserved disulphide that is essential for function. Here, we have examined the effects of sulphydryl redox reagents on [³H]nicotine binding to chick brain nAChR immunoisolated with the monoclonal antibody mAb35. The disulphide reducing agent, dithiothreitol (DTT), inhibited [³H]nicotine binding [50% inhibitory concentration (IC₅₀) = 146 μM] but this effect was reversed (93±1.5%) by subsequent reoxidation with 1 mM dithio-bis(nitrobenzoic acid) (DTNB). The trivalent arsenical, p -aminophenyl dichloroarsine (APA), which reacts with pairs of spatially close sulphydryls, was a potent inhibitor of reoxidation by DTNB (IC₅₀= 35 nM). However, application of the 'anti-arsenical', 2,3-dimercaptopropane sulphonic acid (DMPS), restored agonist binding after APA treatment (50% effective concentration = 120 μM). Paradoxically, DMPS was also found to be a potent oxidizing agent of these receptors. Affinity alkylation of reduced nAChRs with bromoacetylcholine (BAC; 100 μM) irreversibly blocked nicotine binding (>90%). We propose (but have not proven) that APA interacts with the cysteines homologous to Cys192–193 in Torpedo AChRs, since APA pretreatment of reduced neuronal receptors protected against irreversible BAC alkylation, as shown by subsequent reversal of DMPS (2 mM; 20 min). This study illustrates the potent and reversible nature of the arsenical's covalent interaction with an isolated nAChR and suggests that modified arsenicals could be useful nAChR probes.     

Evidence For Two Neurochemical Divisions in the Human Nucleus Accumbens

The neurochemical anatomy of the human nucleus accumbens was studied by comparing the distributional patterns of [³H]DAMGE (μ opioid receptor), [³H]bremazocine (κ opioid receptor), [³H]SCH-23390 (D₁-like dopamine receptor), [³H]7-OH-DPAT (D₃ dopamine receptor) binding, preproenkephalin mRNA and acetylcholinesterase activity in sections of post mortem human striatum. Our results demonstrate the presence of at least two neurochemically distinct divisions within the human nucleus accumbens, which may be homologous to the 'shell'and'core'divisions of the nucleus as found in the rat.     

Gp120 can revert antagonism at the glycine site of NMDA receptors mediating GABA release from cultured hippocampal neurons

The effects of the human immunodeficiency virus type 1 envelope protein gp120 on the release of GABA elicited by N-methyl-D-aspartate (NMDA) from rat hippocampal neurons in primary culture has been investigated. NMDA (1–300 μM) increased in a concentration-dependent manner (EC₅₀ = 37.9 ± 12 μM) the release of [³H]-GABA. The effect of 100 μM NMDA was prevented by 30 μM of the GABA transport inhibitor N-(4,4-diphenyl-3-butenyl)guvacine (SKF 100330A). Glycine (10 μM) or gp120 (0.01 μM) affected neither the basal nor the NMDA-evoked [³H]-GABA release. The NMDA (100 μM)-evoked release was prevented by 5,7-dichloro-kynurenic acid (5,7-DCKA), a selective antagonist at the glycine site of the NMDA receptor, in a concentration-dependent manner (IC₅₀ ≃ 0.3 μM). Glycine (3–10 μM) or gp120 (0.003–0.01 μM) produced reversal of the 5,7-DCKA antagonism in a way that suggested competition at a same site; gp120 was at least 3 orders of magnitude more potent than glycine. It is suggested that gp120 may mimic glycine at NMDA receptors. J. Neurosci. Res. 49:732–738, 1997. © 1997 Wiley-Liss, Inc.     

Plasticity of &mgr; and delta Opioid Receptors in the Superficial Dorsal Horn of the Adult Rat Spinal Cord Following Dorsal Rhizotomies: A Quantitative Autoradiographic Study

The aim was to study the regulation of μ and δ opioid binding sites in the superficial layers (laminae I–II) of the dorsal horn of the adult rat spinal cord 1, 2, 4 and 12 weeks after unilateral dorsal rhizotomies of various extents. Using quantitative autoradiography and highly selective tritiated opioid ligands, we have shown that the decrease in [³H]Tyr*- d -Ala-Gly-NMe-Phe-Gly-ol ([³H]DAMGO) (μ sites) and [³H]Tyr*- d -Thr-Gly-Phe-Leu-Thr ([³H]DTLET) (δ sites) binding in the side ipsilateral to the lesion as compared to the intact side is related to the number of dorsal roots cut. In the segment central to the lesion, 1 week after the lesion, ipsilateral/contralateral side binding ratios for [³H]DAMGO were 0.70, 0.49, 0.36 and 0.25 when 1, 3, 5 and 7 roots respectively were sectioned. For [³H]DTLET, the ratios were 0.71, 0.54, 0.42 and 0.39. The time-related analysis of binding ratios showed that, in partially deafferented spinal segments after long-term deafferentation (12 weeks postlesion) there were greater numbers of μ and δ binding sites than in cases of short-term deafferentation (1–2 weeks). By contrast, in spinal segments considered as completely deafferented, there was no difference in the remaining μ and δ binding sites at 12 weeks as compared to 1 week postlesion. Consequently, it is deduced that the partial recovery of μ and δ binding observed after long-term partial deafferentation could be associated with neuronal plasticity (probably collateral sprouting) of fine diameter primary afferent fibres arising from intact dorsal roots.     

Copper Modulation of NMDA Responses in Mouse and Rat Cultured Hippocampal Neurons

The effect of Cu²⁺ on NMDA receptors was studied in cultured mouse and rat hippocampal neurons using whole-cell patch-clamp and a fast perfusion system. Analysis of the Cu²⁺ concentration-response curve for inhibition of NMDA-induced currents suggests that free Cu²⁺ directly inhibits NMDA receptors with an IC₅₀ of 0.27 μM. Cu²⁺ was ineffective in blocking NMDA receptor activity when complexed with NMDA or glycine; NMDA-Cu²⁺ and glycine-Cu²⁺ complexes acted as agonists of similar potency to the free amino acids. The inhibition by Cu²⁺ (10–100 μM) of responses to 10 μM NMDA was essentially voltage-independent. The onset of inhibition by 100 μM Cu²⁺ of responses to 2 FM glutamate acting at NMDA receptors was significantly faster than NMDA receptor deactivation evoked by a sudden decrease in the concentration of glycine or glutamate, or of both agonists. This suggests that CU²⁺ acts as a non-competitive antagonist, and does not directly interfere with the binding of glutamate or glycine to their recognition sites on the NMDA receptor complex. In the absence of NMDA the apparent association rate constant for binding of Cu²⁺ to NMDA receptors, calculated from the rate of onset of block by Cu²⁺ of test responses to NMDA, was 19 times slower than in the presence of 30 μM NMDA, suggesting that Cu^z+ interacts preferentially with agonist-bound receptors. Our results show that Cu²⁺ is a potent inhibitor of NMDA receptor-mediated responses.     

The effect of acute hypoglycemia on the cerebral NMDA receptor in newborn piglets

The effects of acute insulin-induced hypoglycemia on the cerebral NMDA receptor in the newborn were examined by determining [³H]MK-801 binding as an index of NMDA receptor function in 6 control and 7 hypoglycemic piglets. In hypoglycemic animals, the glucose clamp technique with constant insulin infusion was used to maintain a blood glucose concentration of 1.2 mmol/l for 120 min before obtaining cerebral cortex for further analysis; controls received a saline infusion. Concentrations of glucose, lactate, ATP, and PCr were measured in cortex, and Na⁺,K⁺-ATPase activity was determined in a brain cell membrane preparation. [³H]MK-801 binding was evaluated by: (1) saturation binding assays over the range of 0.5–50 nM [³H]MK-801 in the presence of 100 μM glutamate and glycine; and (2) binding assays at 10 nM [³H]MK-801 in the presence of glutamate and/or glycine at 0, 10, or 100 μM. Blood and brain glucose concentrations were significantly lower in hypoglycemic animals than controls. There was no change in brain ATP with hypoglycemia, but PCr was decreased 80% compared to control (P < 0.05). Na⁺,K⁺-ATPase activity was 13% lower in hypoglycemic animals (P < 0.05). Based on saturation binding data, hypoglycemia had no effect on the number of functional receptors (B_max), but the apparent affinity was significantly increased, as indicated by a decrease in the K_d (dissociation constant) from the control value of 8.1 ± 1.6 nM to 5.5 ± 2.1 nM (P < 0.05). Augmentation of [³H]MK-801 binding by glutamate and glycine alone or in combination was also significantly greater in the hypoglycemic animals. These data suggest that acute hypoglycemia may enhance the excitotoxic effects of glutamate in the newborn.     

N-methyl-D-aspartate autoreceptors respond to low and high agonist concentrations by facilitating, respectively, exocytosis and carrier-mediated release of glutamate in rat hippocampus

Presynaptic NMDA autoreceptors regulating glutamate release have rarely been investigated. High-micromolar N-methyl-D-aspartate (NMDA) was reported to elicit glutamate release from hippocampal synaptosomes in a Ca(2+)-independent manner by reversal of excitatory amino acid transporters. The aim of this work was to characterize excitatory amino acid release evoked by low-micromolar NMDA from glutamatergic axon terminals. Purified rat hippocampal synaptosomes were prelabelled with [(3)H]D-aspartate ([(3)H]D-ASP) and exposed in superfusion to varying concentrations of NMDA in the presence of 1 microM glycine. The release of [(3)H]D-ASP and also that of endogenous glutamate provoked by 10 microM NMDA were external Ca(2+) dependent and sensitive to the NMDA channel blocker MK-801 but insensitive to the glutamate transporter inhibitor DL-TBOA, which, on the contrary, prevented the Ca(2+)-independent release evoked by 100 microM NMDA. The NMDA (10 microM) response was blocked by 1 nM Zn(2+) and 1 microM ifenprodil, compatible with the involvement of a NR1/NR2A/NR2B assembly, although the presence of two separate receptor populations, i.e., NR1/NR2A and NR1/NR2B, cannot be excluded. This response was strongly antagonized by submicromolar (0.01-1 microM) concentrations of kynurenic acid and was mimicked by quinolinic acid (1-100 microM) plus 1 microM glycine. Finally, the HIV-1 protein gp120 potently mimicked the NMDA co-agonists glycine and D-serine, being significantly effective at 30 pM. In conclusion, glutamatergic nerve terminals possess NMDA autoreceptors mediating different types of release when activated by different agonist concentrations: low-micromolar glutamate would potentiate glutamate exocytosis, whereas higher glutamate concentrations would also provoke carrier-mediated release.     

7-Chlorokynurenate Ameliorates Neuronal Injury Mediated by HIV Envelope Protein gp120 in Rodent Retinal Cultures

Prior studies with in vitro model systems have suggested that at least part of the neurological manifestations of AIDS may stem from neuronal injury involving the HIV-1 coat protein gp120. This form of neuronal damage is most probably mediated indirectly by a complex set of cellular interactions among macrophages, astrocytes, and neurons, resulting in a final common pathway of overstimulation of N -methyl- d -aspartate (NMDA) receptors. We studied the neuroprotective effect from gp120-induced neuronal injury of an antagonist of the glycine site of the NMDA receptor, 7-chlorokynurenate. In identified rat retinal ganglion cells in culture, we found that 50 μM 7-chlorokynurenate significantly abrogated the injury engendered by 20 pM gp120. Addition of 300 μM exogenous glycine prevented this protective effect of 50 μM 7-chlorokynurenate. These data suggest that glycine site antagonists of the NMDA receptor may have therapeutic potential for ameliorating neuronal damage associated with gp120.     

Effects of the Anticonvulsant Losigamone and Its Isomers on the GABAA Receptor System

Summary: We conducted in vitro studies to clarify the possible involvement of GABA, receptor-mediated processes in the anticonvulsant effects of losigamone and its optical isomers AO-242 (+losigamone) and AO-294 (-losigamone). In binding experiments with cortical and cerebellar membrane preparations of rat brain, ≤100 μM losigamone did not affect the specific binding of [³H]GABA, [³H]flunitrazepam, or [³⁵S]t-butyl-bicyclo-phosphorothionate (TBPS) to their receptors. Losigamone, however, in concentrations of 10-8-10-5 M, stimulated ³⁶Cl influx into spinal cord neurons in the absence of exogenous GABA. This effect was inhibited by the GABA antagonists bicuculline (BIC) and picrotoxin (PIC). Losigamone 10^-5 M potentiated the effect of a suboptimal concentration of exogenous GABA M on 3³⁶Cl influx. Both isomers of losigamone likewise stimulated 36Cl influx into spinal cord neurons, and these effects were similarly antagonized by BIC and PIC. Losigamone and its optical isomers AO-294 and AO-242 antagonized potassium-induced hyperexcitability in rat hippocampal slices concentration dependently. There were no clear differences in the potencies of losigamone, AO-242, or AO-294. However, AO-294 and AO-242 differed significantly in their ability to suppress TBPS- induced hyperexcitability of hippocampal slices. Such observations demonstrate that although losigamone does not bind to GABA, benzodiazepine (BZD) or PIC binding sites of the neuronal chloride channel, it is capable of stimulating ³⁶Cl influx in the spinal cord neurons by a GABA-sensitive mechanism and at a side distant from the GABA channel.     

Neurosteroids: Expression of Functional 3β-Hydroxysteroid Dehydrogenase by Rat Sensory Neurons and Schwann Cells

Steroids which are synthesized within the nervous system, such as progesterone, have been termed 'neurosteroids'. Levels of progesterone are much larger in peripheral nerves of rats and mice than in plasma, and persist after removal of the steroidogenic endocrine glands. Schwann cells are a source of progesterone: when isolated from embryonic dorsal root ganglia, they can synthesize progesterone from pregnenolone, the obligate precursor of all steroids. Locally produced progesterone has been shown to play an important role in myelination of peripheral nerve. We show here that sensory neurons from embryonic dorsal root ganglia also express 3β-hydroxysteroid dehydrogenase and can convert [³H]pregnenolone to [³H]progesterone. Moreover, when cultured under different conditions and incubated for 24 h in the presence of 100 nM [³H]pregnenolone, they produce 5–10 times more [³H]progesterone than Schwann cells. The conversion of pregnenolone to progesterone by neurons is further increased by a diffusible factor produced by Schwann cells. Sensory neurons can also metabolize progesterone to 5α-dihydroprogesterone, but unlike Schwann cells, they do not produce 3α,5α-tetrahydroprogesterone, a potent positive allosteric modulator of γ-aminobutyric acid type A receptors. We also show that cells isolated from the adult nervous system still have the capacity to convert [³H]pregnenolone to progesterone and its 5α-reduced metabolites: neurons and Schwann cells purified from dorsal root ganglia of 6 week old male rats show a similar pattern of pregnenolone metabolism to cells isolated from 18 day old embryos. These findings further support the important role of progesterone in the development and regeneration of the peripheral nervous system.     

Postischemic changes of [3H]nimodipine and [3H]ryanodine binding in the gerbil striatum and hippocampus

Sequential alterations of [³H]nimodipine and [³H]ryanodine binding in gerbils were investigated in selectively vulnerable regions, such as the striatum and hippocampus, 1 h to 7 days after 10 min of transient cerebral ischemia. [³H]Nimodipine binding showed no significant changes in the striatum and hippocampus up to 48 h after ischemia. Seven days after ischemia, however, a severe reduction in [³H]nimodipine binding was observed in the dorsolateral striatum, hippocampal CA1 (stratum oriens, stratum pyramidale and stratum radiatum) and hippocampal CA3 sector. On the other hand, [³H]ryanodine binding showed a significant increase in the hippocampus 1 h after ischemia. Five hours after ischemia, a significant reduction in [³H]ryanodine binding was observed only in the hippocampal CA1 sector. Thereafter, the striatum and hippocampus showed no significant alterations in [³H]ryanodine binding up to 48 h after ischemia. After 7 days, a marked reduction in [³H]ryanodine binding was observed in the striatum and hippocampus which were particularly vulnerable to ischemia. These results demonstrate that postischemic alteration in [³H]nimodipine and [³H]ryanodine binding is produced with different processes in the hippocampus. They also suggest that the mechanism for striatal cell damage caused by transient cerebral ischemia may, at least in part, differ from that for hippocampal neuronal damage. Furthermore, our findings suggest that abnormal calcium release from intracellular stores may play a pivotal role in the development of hippocampal neuronal damage.     

[3H]Nitrendipine Binding to Rabbit Colonic Smooth Muscle

We used [³H] nitrendipine binding to isolated smooth muscle cells and isometric tension studies of muscle strips to characterize the calcium channels from rabbit proximal and distal colon. At 25°C [³H] nitrendipine binding was rapid, saturable, reversible, specific, and linearly proportional to cell number. The affinity of the ligand for its receptor was similar in proximal and distal colon (K_D 129 ± 21 pM and 124 ± 17 pM, respectively). In the proximal colon there were 68,000 receptors per cell, compared to 58,000 receptors per cell in the distal colon (p > .1). The Hill coefficient for nitrendipine was close to unity, suggesting binding to a single receptor. Although nitrendipine and nifedipine competitively inhibited [³H]nitrendipine binding, verapamil did not alter [³H] nitrendipine binding, suggesting the presence of at least two discrete, noninteracting sites for the binding of drugs that block calcium channels. In studies with muscle strips nitrendipine competitively inhibited isometric tension stimulated by both bethanechol and high extracellular potassium concentration. There were no significant differences in response from proximal and distal colon. These results suggest that calcium antagonist binding characteristics to calcium channels are similar in proximal and distal colon, and do not explain previously observed differences in the function of muscle in these tissues.     

Alterations of G-protein Coupling Function in Phosphoinositide Signalling Pathways of Rat Hippocampus by Ischaemic Brain Injury

The activation of membrane-associated phospholipase C is rapidly and transiently induced in the central nervous system by a variety of stimuli. Ischaemic brain injury is one of the situations that leads to a dramatic increase in polyphosphoinositide (PPI) turnover. In this study, stimulation of PPI hydrolysis by glutamate (500 μM) was measured in hippocampal slices from rats up to 21 days after an ischaemic insult of 30 min. Ischaemia was induced using the four-vessel occlusion method. PPI hydrolysis elicited by glutamate was significantly increased in the slices prepared from ischaemic rats 24 h after reperfusion, the accumulation of inositol phosphates (InsP_s) and inositol 1,4,5-trisphosphate (InsP₃) was 614±74% ( n = 8) and 182±11% ( n = 9) of the basal level respectively. This potentiation was also observed 21 days after ischaemia. Hyper-responsiveness to glutamate was also accompanied by an increase in AIF⁻₄-stimulated formation of [³H]inositol phosphates. In addition, global ischaemia did not change either high-affinity [³H]glutamate binding in hippocampal membranes or the stimulation of PPI hydrolysis by carbachol or noradrenaline in hippocampal slices. The present results suggest that the increased responsiveness to glutamate is the result, at least in part, of functional changes at the G-protein level, and may contribute to the pathophysiology of ischaemic brain injury or to the regenerative phenomena that accompany ischaemic damage.     

β-Carbolines: Endogenous Ligands for the Benzodiazepine Receptor

Abstract: The interaction of benzodiazepines (BDZ) and β -carbolines with metal cations was investigated. Δmong the numerous transition metal cations, only four, Co²⁺, Ni², Cu²⁺ and Zn²⁺, specifically affected the receptor binding of [³H]diazepam and [³H] β -carboline-3-carboxylic acid ethyl ester ( β -CCE). The effects of these cations on [³H]diazepam binding were exactly opposite to those on [³H] β -CCE binding. An intraperitoneal injection of β -carboline-3-carboxylic acid methyl ester ( β -CCM) produced spike discharges and β-CCM induced spike discharges were prevented by an intramuscular injection of BDZ. These findings suggest that β -carbolines could be related to the proposed endogenous ligand and that BDZ might be antagonistic rather than agonistic of the naturally occurring ligand.     

In Vivo Modulation of Sigma Receptor Sites by Calcitonin Gene-related Peptide in the Mouse and Rat Hippocampal Formation: Radioligand Binding and Electrophysiological Studies

Possible interactions between sigma (σ) receptor sites and calcitonin gene-related peptides (CGRP) were investigated using receptor subtype-related analogues and fragments in in vivo [³H](+)SKF 10 047/σ binding in the hippocampus, and electrophysiological recording of the N -methyl-D-aspartate (NMDA)-induced activation of CA3 pyramidal neurons, two well-established σ assays. In both paradigms, CGRP and the agonist [Cys(ACM)^2,7]hCGRPα modulated σ systems. In vivo binding experiments demonstrated that CGRP and [Cys(ACM)^2,7]hCGRPα inhibited 25–40% of specific [³H](+)SKF 10 047 labelling in the mouse hippocampal formation while the purported antagonist hCGRP_8–37 was inactive. The specificity of this modulation was demonstrated further by the lack of effect of other vasoactive peptides, including the atrial natriuretic peptide, substance P, and its N-terminal fragment, substance P_1–7. In the CA3 subfield of the rat dorsal hippocampus, hCGRPα decreased (up to 61%) the NMDA-induced activation of the pyramidal neurons. Conversely, the linear analogue [Cys(ACM)^2,7]hCGRPα enhanced (by 85%) the NMDA-induced activation of CA3 pyramidal neurons, while the antagonistic fragment hCGRP_8–37 had no effect. Haloperidol, a high-affinity σ receptor ligand, inhibited by 90% the in vivo [3H](+)SKF 10 047 labelling, and prevented the modulation of the NMDA-induced activation by hCGRPα and [Cys(ACM)^2,7]hCGRPα. It thus appears that CGRP can modulate σ-related systems in the hippocampal formation.     

Death of cultured human neuroblastoma cells induced by HIV-1 gp120 is prevented by NMDA receptor antagonists and inhibitors of nitric oxide and cyclooxygenase

The cytotoxic effects of the human immunodeficiency virus type 1 (HIV-1) coat protein gp120 were studied in human CHP100 neuroblastoma cell cultures. Incubation of neuroblastoma cultures with gp120 (1 pM-10 nM) induces cell death which is not concentration-related. The significant cell death evoked by 10 pM gp120 was prevented by neutralization of the viral protein with a monoclonal anti-gp120 (IgG) antibody. In addition, gp120-induced cytotoxicity was inhibited by [DL-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid] (CGP37849; 100 μM), [(±)-3R1, 4as1, 6R1, 8aR1-6-(phosphonomethyl) decahydro-isoquinoline-3-carboxylic acid] (LY274614; 100 μM), MK801 (dizocilpine; 200 nM) and 7-chloro kynurenic acid (100 μM), selective antagonists of the NMDA receptor complex; by contrast, (6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 100 μM), a non-NMDA antagonist, was ineffective. Prevention of the lethality elicited by the HIV-1 coat protein was also obtained by incubating neuroblastoma cells with gp120 in Ca²⁺-free medium. The lethal effects induced by gp120 involve activation of L-arginine-nitric oxide (NO) pathway since these were prevented by haemoglobin (10 μM), a NO-trapping agent, and by D-arginine (1 mM), the less active enantiomer of the endogenous precursor of NO synthesis. Cytoprotection was also afforded by N^ω-nitro-L-arginine methyl ester (L-NAME; 200 μM), an inhibitor of NO synthase, and this was reversed by L-arginine (1 mM). Interestingly, indomethacin and flufenamic acid (10 μM), two inhibitors of cyclooxygenase, protected neuroblastoma cells from death induced by gp120. Furthermore, indomethacin prevented the neuroblastoma cell death evoked by exposure of cultures to sodium nitroprusside (SNP; 0.2–1.6 mM), a NO donor. Finally significant cytotoxic effects were observed after incubation of neuroblastoma cells with prostaglandin E₂ (0.1–10 μM). In conclusion, the present data suggest that death of human CHP100 neuroblastoma cells in culture produced by gp120 involves NO and PGE₂ production.     

α7 and non-α7 nicotinic acetylcholine receptors modulate dopamine release in vitro and in vivo in the rat prefrontal cortex

Nicotine enhances attentional and working memory aspects of executive function in the prefrontal cortex (PFC) where dopamine plays a major role. Here, we have determined the nicotinic acetylcholine receptor (nAChR) subtypes that can modulate dopamine release in rat PFC using subtype-selective drugs. Nicotine and 5-Iodo-A-85380 (β2* selective) elicited [³H]dopamine release from both PFC and striatal prisms in vitro and dopamine overflow from medial PFC in vivo . Blockade by dihydro-β-erythroidine supports the participation of β2* nAChRs. However, insensitivity of nicotine-evoked [³H]dopamine release to α-conotoxin-MII in PFC prisms suggests no involvement of α6β2* nAChRs, in contrast to the striatum, and this distinction is supported by immunoprecipitation of nAChR subunits from these tissues. The α7 nAChR-selective agonists choline and Compound A also promoted dopamine release from PFC in vitro and in vivo , and their effects were enhanced by the α7 nAChR-selective allosteric potentiator PNU-120596 and blocked by specific antagonists. DNQX and MK801 inhibited [³H]dopamine release evoked by choline and PNU-120596, suggesting crosstalk between α7 nAChRs, glutamate and dopamine in the PFC. In vivo, systemic (but not local) administration of PNU-120596, in the absence of agonist, facilitated dopamine overflow in the medial PFC, consistent with the activation of extracortical α7 nAChRs by endogenous acetylcholine or choline. These data establish that both β2* and α7 nAChRs can modulate dopamine release in the PFC in vitro and in vivo . Through their distinct actions on dopamine release, these nAChR subtypes could contribute to executive function, making them specific therapeutic targets for conditions such as schizophrenia and attention deficit hyperactivity disorder.