Bio-prospecting endemic Mascarene Aloes for potential neuroprotectants.

The Mascarene Aloes are used in the traditional pharmacopoeia against various ailments including cutaneous diseases and as antispasmodics. Scientific evidence to support these claims is non-existent and mainly based on the scientific repute of A. vera. The antioxidant profile of methanolic leaf extracts of A. purpurea Lam., A. tormentorii (Marais) L. E. Newton & G. D. Rowley, A. lomatophylloides Balf. f., A. macra Haw. and A. vera (L.) Burm. f. was studied using the total antioxidant capacity, copper equivalent and superoxide dismutase assays. In vitro cytotoxicity was evaluated on CAD (Cath.-a-differentiated) neuronal cells by the methyl tetrazolium assay, and the neuroprotective profile was assessed using hydrogen peroxide-induced neurotoxicity with the CAD cells. The aloin and vitexin content were determined by high-performance liquid chromatography with diode-array detection. A. purpurea had the highest aloin content (546.6 nmol/g), while A. tormentorii had the highest vitexin content (67.3 nmol/g). A. macra (concentration <0.1 mg/mL) elicited a 10% cytotoxicity effect on CAD cells while other Mascarene Aloes were not cytotoxic. This study validates the antioxidant and neuroprotective potential of Mascarene Aloes focusing on their aloin and vitexin content that are also present in other reputed medicinal Aloes.

Effects of chronic fluoxetine treatment on anxious behaviour of BALB/c mice in a 3-dimensional maze.

Here we used a 3-dimensional (3D) maze, a modification of the radial maze, to assess the effects of treatment for two weeks with a single daily dose of fluoxetine (20 mg/kg, i.p.) on anxiety in male BALB/c mice. We examined whether anxiolytic effects of fluoxetine can be detected over three daily test sessions. We examined also whether repeated handling associated with chronic treatment interferes with effects of fluoxetine on anxiety responses. The 3D maze comprises nine arms, each connected to an upward inclined bridge radiating from a central platform. In this maze, BALB/c mice cross frequently into the bridges but avoid the arms. This avoidance is used as an index of anxiety. Two separate groups received once a day either saline (SALCH, n = 8) or fluoxetine (FLUCH, n = 8) for 14 days, and up to 30 min before the test during the subsequent 3 days. A third group received saline (SALAC, n = 8) 30 min before the test, once a day for 3 days. SALAC mice did not cross into the arms, and continued this avoidance over 3 sessions. SALCH mice avoided the arms in session 1 whereas FLUCH mice did cross into the arms, and like SALCH mice, increased number of crossings into and time on the arms in subsequent sessions. Fluoxetine evidently had an anxiolytic effect but only in the first session. These results indicate that handling experience decreased fear and anxiety in the mice, which may have masked the anxiolytic effect of fluoxetine in the second and third test sessions.

Overexpression of histamine H4 receptors in the kidney of diabetic rat.

OBJECTIVE AND DESIGN: The renal expression of H1 and H2 receptors has previously been demonstrated, while that of the H4 receptor has been poorly investigated, and thus the aim of this research was to investigate the expression of the H4 receptor in the kidney of diabetic rats. MATERIAL OR SUBJECTS: 24 8-week-old male Wistar rats. TREATMENT: Diabetes was induced in 12 rats by a single intravenous injection of streptozotocin, and animals were killed 6 weeks later. METHODS: Kidneys were collected and processed for quantitative PCR or immunohistochemical analyses. To ascertain the renal topology of the H4 receptor, colocalization experiments were performed with a series of markers. RESULTS: H4 receptor is expressed in healthy rats, although at a very low level, and is strongly upregulated in diabetic animals. Immunohistochemical analysis revealed the highest immune-positivity in the medulla. Colocalization experiments revealed a close overlap in expression topology of the H4 receptor and both Tamm-Horsfall glycoprotein and aquaporin 1 was observed. CONCLUSIONS: The results demonstrate, for the first time, that the H4 receptor is expressed in the kidney mainly by resident renal cells of the loop of Henlé and that this receptor is significantly overexpressed in diabetic animals, thus suggesting a possible role in the pathogenesis of diabetes-associated renal disease.

Brief report: first identification of H4 histamine receptor in healthy salivary glands and in focal sialadenitis in Sjögren's syndrome.

OBJECTIVE: The conventional H(1) and H(2) histamine receptors have >10,000-fold lower avidity for histamine than H(4) histamine receptor, which has been implicated in autoimmune diseases. This study was undertaken to compare H(4) histamine receptor levels in the salivary glands (SGs) of healthy controls with those in the SGs of patients with primary Sjögren's syndrome (SS). METHODS: H(4) histamine receptor messenger RNA (mRNA) was analyzed using real-time quantitative polymerase chain reaction, and the receptor protein was examined using immunostaining. Effects of the H(4) histamine receptor agonist ST-1006 on cytokine synthesis by human SG (HSG) cells were analyzed using xMAP technology and enzyme-linked immunosorbent assay. RESULTS: Healthy SGs contained H(4) histamine receptor mRNA. The receptor protein was localized to the acinar and ductal epithelial cells. H(4) histamine receptor agonist stimulated HSG cells to produce the cytokines interleukin-8 and vascular endothelial growth factor. SS patients had low H(4) histamine receptor levels. CONCLUSION: H(1) and H(2) histamine receptor antagonists are not effective in the treatment of autoimmune diseases. However, such antagonists do not affect the newly discovered H(4) histamine receptor. Dendritic cells and lymphocytes are nonprofessional histamine-producing cells, which produce histamine at 100-1,000-fold lower rates than mast cells do. Saliva contains only 0.31-12.4 ng/ml histamine, which is too low to stimulate H(1) or H(2) histamine receptor, but stimulates H(4) histamine receptor half maximally. Our findings show that H(4) histamine receptor is strongly expressed in tubuloacinar SG cells, which emphasizes the role of these cells in the pathogenesis of SS.

Immunological identification of the mouse H4 histamine receptor on spinal cord motor neurons using a novel anti-mouse H4R antibody.

In this report, we describe the development of the first anti-mouse H(4) receptor antibody. Utilising this new immunological probe, new evidence is provided for oligomeric mH(4)Rs and the presence of H(4) receptors on a subpopulation of murine motor neurons. This expands the cell types where the H(4) receptor is expressed in the mammalian CNS.

A new model for the study of high-K(+)-induced preconditioning in cultured neurones: role of N-methyl-d-aspartate and alpha7-nicotinic acetylcholine receptors.

Spreading depression (SD), whether elicited by local application of high K(+) medium to the cortical surface or by other stimuli, can increase the brain's tolerance to a subsequent, severe ischaemic insult in vivo, a phenomenon termed preconditioning. Herein, we have developed and validated a robust in vitro protocol for high-K(+)-preconditioning of cultured neurones. This new model is especially appropriate to unravel the molecular mechanisms underlying neuronal preconditioning and subsequent ischaemic tolerance. With this new, optimised preparation, preconditioning was found to be dependent upon culture day in vitro, cell density, K(+) concentration and duration of treatment. Finally, preconditioning was shown to be dependent upon N-methyl-d-aspartate (NMDA), CAM-kinase II signalling and alpha7-nicotinic (alpha7 nACh) receptor function, which is analogous to in vivo preconditioning induced by various stimuli.

Emotional responses and memory performance of middle-aged CD1 mice in a 3D maze: effects of low infrared light.

Non-thermal near infra-red (IR) has been shown to have many beneficial photobiological effects on a range of cell types, including neurons. In the present study, a pretreatment with a daily 6 min exposure to IR1072 for 10 days yielded a number of significant behavioral effects on middle-aged female CD-1 mice (12-months) tested in a 3D-maze. Middle-aged mice show significant deficits in a working memory test and IR treatment reversed this deficit. Interestingly, the IR treated middle-aged group despite making less memory errors than sham middle-aged group spent longer time in different parts of the maze than both the young group (3-months) and sham-middle-aged group (12-months). Young mice appeared more anxious than middle-aged mice in the first sessions of the test. Exposure to IR appeared to have no significant effects upon exploratory activity or anxiety responses. However, it elicited significant effects on working memory, with the IR middle-aged mice being more considerate in their decision making, which results in an overall improved cognitive performance which is comparable to that of young CD-1 mice. The present study describes a novel method for assessing emotional responses and memory performance in a 3D spatial navigation task and demonstrates the validity of our new all-in-one test and its sensitivity to ageing and non-invasive beneficial IR treatment.

Detailed analysis of the behavior and memory performance of middle-aged male and female CD-1 mice in a 3D maze.

Fifty percent of CD-1 mice from both sex die by the end of 2 years. The survival rate is higher in females than in males. This high mortality rate is associated to the high susceptibility of this strain of mice to some immuno-pathologies and the high incidence of systemic amyloidosis. It is therefore possible that premature cognitive deficits can be observed in CD-1 mice. In the present study, we describe a novel method for assessing emotional responses and memory performance of young (4 months) and middle-aged (12 months) CD-1 mice of both sexes in a 3D spatial navigation task. Animals are introduced to the maze without preliminary habituation and trained in a working memory test. As expected CD-1 mice have a low number of entries to arms on their first exposure to the maze which confirm our previous report on the anxious trait of this strain compared to C57/BL6 mice. The measure of arm/bridge ratio suggests that anxiety induced by exposure to the maze persists much longer in middle-aged male mice compared to middle-aged female mice and compared to both young male and female mice. The measure of memory revealed that young female mice made significantly less arm repeats and more unique arm visits before first arm repeat than middle-aged female and male mice. There are also significant differences between young female and young male mice with the former committing fewer errors than the latter.

Are benzodiazepines really anxiolytic? Evidence from a 3D maze spatial navigation task.

The effects of diazepam and chlordiazepoxide were assessed in a 3D maze which is a modification of an 8-arm radial maze. Each arm of the maze is attached to a bridge radiating from a central platform. Animals exposed for the first time to the maze do not venture beyond the line that separate a bridge from an arm. The prime criteria set for an anxiolytic effect is whether mice would increase the frequency of entries onto arms and increase arm/bridge entries ratio. C57 mice readily cross the line on first exposure and make more than 8 arm visits onto arms on second exposure, while other strains (CD-1 and Balb/c) hold back and rarely cross the line on first exposure and require more sessions to make more than 8 arm entries. An anxiolytic drug is expected to encourage intermediate (CD-1) and high (Balb/c) anxiety mice to adventure onto the arms of the maze and make more visits to the arms to comparable levels seen with low anxiety c57 mice. In the present report, administration of different doses of diazepam (0.625, 1.25, 2.5 and 5 mg kg(-1) i.p.) and chlordiazepoxide (5, 10 and 15 mg kg(-1) i.p.) did not reduce anxiety in animals, with the lowest dose of diazepam increasing motor activity in Balb/c and increasing anxiety in c57 mice while the highest doses of both diazepam (2.5 and 5 mg kg(-1) i.p.) and chlordiazepoxide (15 mg kg(-1) i.p.) induced mild sedation. Our results raise some concerns about the methodological foundations in the current assessment of anxiety and anxiolytic compounds both in animal and human studies.

Medicinal Mascarene Aloes: An audit of their phytotherapeutic potential.

A phytochemical and biological investigation of the endemic Mascarene Aloes (Aloe spp.), including A. tormentorii (Marais) L.E.Newton & G.D.Rowley, A. purpurea Lam, A. macra Haw., A. lomatophylloides Balf.f and A. vera (synonym A. barbadensis Mill.), which are used in the traditional folk medicine of the Mascarene Islands, was initiated. Methanolic extracts of the Aloes under study were analysed using high resolution LC-UV-MS/MS and compounds belonging to the class of anthraquinones, anthrones, chromones and flavone C-glycosides were detected. The Mascarene Aloes could be distinguished from A. vera by the absence of 2″-O-feruloylaloesin and 7-O-methylaloeresin. GC-MS analysis of monosaccharides revealed the presence of arabinose, fucose, xylose, mannose and galactose in all the Mascarene Aloes and in A. vera. The crude extracts of all Aloes analysed displayed antimicrobial activity against Bacillus cereus, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. Only extracts of A. macra were active against P. aeruginosa and Klebsiella pneumoniae, while none of the Aloe extracts inhibited Propionibacterium acnes. A. macra displayed anti-tyrosinase activity, exhibiting 50% inhibition at 0.95mg/mL, and extracts of A. purpurea (Mauritius) and A. vera displayed activity in a wound healing-scratch assay. In vitro cytotoxicity screening of crude methanolic extracts of the Aloes, using the MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) showed that only A. purpurea (Réunion) elicited a modest toxic effect against HL60 cells, with a percentage toxicity of 8.2% (A. purpurea-Réunion) and none of the Aloe extracts elicited a toxic effect against MRC 5 fibroblast cells at a concentration of 0.1mg/mL. Mascarene Aloe species possess noteworthy pharmacological attributes associated with their rich phytochemical profiles.

Slope climbing challenges, fear of heights, anxiety and time of the day.

When exposed to an unfamiliar open space, animals experience fear and attempt to find an escape route. Anxiety emerges when animals are confronted with a challenging obstacle to this fear motivated escape. High anxiety animals do not take risks; they avoid the challenge. The present experiments investigated this risk avoidant behavior in mice. In experiment 1, BALB/c, C57BL/6J and CD-1 mice were exposed to a large platform with downward inclined steep slopes attached on two opposite sides. The platform was elevated 75 and 100cm from the ground, in a standard (SPDS) and in a raised (RPDS) configuration, respectively. In experiment 2, the platform was elevated 75cm from the ground. Mice had to climb onto a stand at the top of upward inclined slopes (SPUS). In experiment 3, BALB/c mice were exposed to SPDS with steep or shallow slopes either in early morning or in late afternoon. In all 3 test configurations, mice spent more time in the areas adjacent to the slopes than in the areas adjacent to void, however only C57BL/6J and CD-1 crossed onto the slopes in SPDS, and crossed onto the stands in SPUS whereas BALB/c remained on the platform in SPDS and explored the slopes in SPUS. Elevation of the platform from the ground reduced the crossings onto the slopes in C57BL/6J and CD-1, and no differences were observed between BALB/c and C57BL/6J. BALB/c mice demonstrated no difference in anxiety when tested early morning or late afternoon; they crossed onto shallow slopes and avoided the steep one.

Models of anxiety: responses of rats to novelty in an open space and an enclosed space.

Exposure to novelty has been shown to induce anxiety responses in a variety of behavioural paradigms. The purpose of the present study was to investigate whether exposition of naïve rats to novelty would result in a comparable or a different pattern of responses in an open space versus enclosed space with or without the presence of an object in the centre of the field. Lewis and Wistar rats of both genders were used to illustrate and discuss the value and validity of these anxiety paradigms. We examined a wide range of measures, which cover several aspects of animals' responses. The results of this study revealed significant differences between the behaviour of animals in an open space and in the enclosed space. It also revealed significant differences in animal's responses to the presence and absence of an object in the open space and in the enclosed space. In the enclosed space, rats spent most of their time in the outer area with lower number of exits and avoided the object area except when there was an object, while in the open space rats displayed frequent short duration re-entries in the outer area and spent longer time in the object area in presence of an object. The time spent in the inner area (away from the outer area and the object area) was significantly longer and the number of faecal boli was significantly higher in the open space than in the enclosed space. In the present report, we will discuss the fundamental differences between enclosed space and open space models, and we will examine some methodological issues related to the current animal models of human behaviour in anxiety. In the enclosed space, animals can avoid the potential threat associated with the centre area of a box and chose the safety of walls and corners, whereas, in the open space animals have to avoid every parts of the field from which there was no safe escape. The response of animals to novelty in an open space model appears more relevant to anxiety than in an enclosed space. The present studies revealed no correlations between the measures of behaviour in enclosed space and the measures of behaviour in open space, which suggest that these two models do not involve the same construct. Our results suggest that the enclosed space model involves avoidance responses while the open space model involves anxiety responses. The open space model can be very useful in understanding the underlying neural mechanisms of anxiety responses, and in assessing the effects of potential anxiolytic drugs.

Models of anxiety: responses of mice to novelty and open spaces in a 3D maze.

The present report describes the emotional responses of different strains of mice to exposure to a novel open space model of anxiety using a 3D spatial navigation task. The 3D maze is modification of the radial maze with flexible arms that can be raised above or lowered below the horizontal level of a central platform. To access the arms animals need to cross a bridge linking the arms to the central platform. In this model, mice are exposed to novelty in an unfamiliar open space setting with no safe alternative. Fear from novelty is compounded with the need to explore. The drive to escape and the drive to approach are intermingled making this open space model radically different from the current models of anxiety which provide animals with the choice between safe and anxiogenic spaces. In a series of experiments, we examined the behaviour of different groups of mice from C57, C3H, CD1 and Balb/c strains. In the first experiment, different groups of C57 mice were tested in one of the three arms configurations. In the second experiment, C57 mice were compared to C3H mice. In the third experiment, C57 mice were compared to CD1 and Balb/c mice in the raised arm configuration over three successive sessions. In the fourth experiment, we examined the behaviour of C57 mice in the lowered arm configuration with an open and an enclosed central. In the final experiment, we examined the difference between C57 and C3H mice of both genders. Using several spatio-temporal parameters of the transition responses between central platform, bridges and arms, we have been able to show consistent results demonstrating significant differences between C57 and C3H mice, and between Balb/c and both C57 and CD1 mice. C3H appear more anxious than C57 mice, and Balb/c mice seem more anxious than C57 and CD1 mice. We also observed significant differences between sexes in C3H mice but not in C57 mice. C3H male mice appear more anxious than C3H female mice and than both C57 male and female mice. In the lowered arm configuration with an enclosed central platform, C57 mice took longer time to make a first entry to an arm, made more visits to bridges before first entry to an arm and required longer time between re-entries to arms, spent longer time on the central platform and shorter time on arms compared to mice in the other arm configurations. They also made frequent entries to the centre and bridges compared to mice in the lowered arm with an open central platform. These results demonstrate not only the sensitivity of the parameters of the test but also the consistencies and concordances of the results which make this 3D maze a valuable new tool in the study of the underlying neural mechanisms of anxiety responses in addition to learning and memory, and in assessing the effects of potential anxiolytic drugs. In this report we examine methodological issues related to the design of animal behavioural paradigms and question the value and the construct validity of the current models of human anxiety.

Altered GABAA α5 subunit expression in the hypothalamic paraventricular nucleus of hypertensive and pregnant rats.

A characteristic of both hypertension and pregnancy is increased sympathetic nerve activity. The level of sympathetic activation is determined, in part, by a tonic GABAergic inhibition arising from the hypothalamic paraventricular nucleus (PVN). In hypertension, decreases in GABAergic inhibition and increases in glutamatergic excitation within the PVN contribute to this sympatho-excitation. In late-term pregnancy however, the sympatho-excitation appears to be mediated by decreases in GABAergic inhibition only. This study examined whether changes in subunit expression for GABAA receptors in the PVN could provide a molecular basis for the sympatho-excitation characteristic of hypertension and pregnancy. Hypertension and pregnancy were accompanied by significant decrease in the GABAA receptor α5 subunit in the PVN. We suggest that decreases in the α5 subunit of the GABAA receptor may be important in mediating the sympatho-excitation observed in both hypertension and pregnancy.

Pre-training in a radial arm maze abolished anxiety and impaired habituation in C57BL6/J mice treated with dizocilpine.

Familiarity can imply a reduction of fear and anxiety, which may render learning and memory performance insensitive to NMDA receptor antagonism. Our previous study indicates that MK-801 (dizocilpine), NMDA antagonist, increased anxiety and prevented the acquisition of a spatial memory task. Here, we examined whether MK-801 will produce anxiety in mice that were familiar with the test environment. Male C57BL/6J mice were exposed, one session a day for 7days, to a 3D maze, which consisted of nine arms attached to upward inclined bridges radiating from a nonagonal platform. In this maze, high anxiety mice avoid the arms in the first sessions. One group of mice received saline (SAL) while a second group received MK-801 (MKD1), both on day one. A third group received saline in the first 3 sessions, and MK 801 in subsequent sessions (MKD4). Saline and MK-801 (0.1mg/kg) were administered intraperitoneally 30min before the test. MKD4 mice demonstrated an increase in bridge and arm visits, and reached arm/bridge entries ratio close to 1 in session 5. SAL mice also crossed frequently onto the arms, and reached a comparable ratio, but this was achieved with a lower number of arm visits. MKD1 mice demonstrated a reduced number of arm visits in each session compared to SAL and MKD4 mice. Dizocilpine produced anxiety in mice treated from day 1 of the test, but not in those treated from day 4. It also impaired habituation in animals familiar with the test environment; it produced sustained non-habituating hyperactivity.

Radioligand binding studies of caloporoside and novel congeners with contrasting effects upon [35S] TBPS binding to the mammalian GABA(A) receptor.

Caloporoside is a natural active fungal metabolite, which was isolated from Caloporous dichrous and was described to exhibit antibacterial, antifungal and phospholipase C inhibitory activity. We have previously reported evidence that related beta-linked compounds, lactose and octyl-beta-d-mannoside, bind and functionally modulate rodent GABA(A) receptors, respectively. We have characterized the binding pharmacology of synthetic caloporoside and two further congeners, 2-hydroxy-6-([(16R)-(beta-d-mannopyranosyloxy)heptadecyl]) benzoic acid and octyl-beta-d-glucoside on GABA(A) receptors using a [35S]-t-butylbicyclophosphoorothionate (TBPS) radioligand binding assay. Caloporoside and 2-hydroxy-6-([(16R)-(beta-d-mannopyranosyloxy)heptadecyl]) benzoic acid produced concentration-dependent complete inhibition of specific [35S] TBPS binding with overall apparent IC50 values of 14.7+/-0.1 and 14.2+/-0.1 microM, respectively. In contrast, octyl-beta-d-glucoside elicited a concentration-dependent stimulation of specific [35S] TBPS binding (E(max)=144+/-4%; EC50=39.2+/-22.7 nM). The level of stimulation was similar to that elicited by diazepam (E(max)=147+/-6%; EC50=0.8+/-0.1 nM), and was occluded by GABA (0.3 microM). However, the three test compounds failed to elicit any significant effect (positive or negative) upon [3H] flunitrazepam or [3H] muscimol binding, indicating that they did not bind directly, or allosterically couple, to the benzodiazepine or agonist binding site of the GABA(A) receptor, respectively. The constituent monosaccharide, glucose, and both the closely related congeners octyl-beta-d-glucoside or hexyl-beta-d-glucoside have no significant effect upon [35S] TBPS binding. These data, together, provide strong evidence that a beta-glycosidic linkage and chain length are crucial for the positive modulation of [35S] TBPS binding to the GABA(A) receptor by this novel chemical class.

Characterization of the binding of two novel glycine site antagonists to cloned NMDA receptors: evidence for two pharmacological classes of antagonists.

The potency of two novel glycine site antagonists, GV150,526A and GV196,771A, was assessed by their ability to inhibit the binding of [(3)H]-MDL105,519 to cell homogenates prepared from mammalian cells transfected with either NR1-1a, NR1-2a, NR1-1a/NR2A, NR1-1a/NR2B, NR1-1a/NR2C or NR1-1a/NR2D NMDA receptor clones. The inhibition constants (K(i)s) for GV150,526A displacement of [(3)H]-MDL105,519 binding to either NR1-1a or NR1-2a expressed alone were not significantly different and were best fit by a one-site binding model. GV150,526A inhibition to NR1-1a/NR2 combinations was best fit by a two-site model with the NR1-1a/NR2C having an approximate 2 - 4 fold lower affinity compared to other NR1-1a/NR2 receptors. The K(i)s for GV196,771A displacement of [(3)H]-MDL105,519 binding to NR1-1a, NR1-2a and all NR1-1a/NR2 combinations was best fit by a two-site binding model. There was no significant difference between the K(i)s for the binding to NR1-1a and NR1-2a; NR1-1a/NR2A receptors had an approximate 4 fold lower affinity for GV196,771A compared to other NR1-1a/NR2 combinations. The K(i)s for both GV150, 526A and GV196,771A for the inhibition of [(3)H]-MDL105,519 binding to membranes prepared from adult rat forebrain were determined and compared to the values obtained for binding to cloned NMDA receptors. The K(i)s for a series of glycine site ligands with diverse chemical structures were also determined for the inhibition of [(3)H]-MDL105,519 binding to NR1-1a/NR2A receptors. L689,560 displayed similar binding characteristics to GV150,526A. It is suggested that glycine site antagonists may be divided into two classes based on their ability to distinguish between NR1 and NR1/NR2 receptors with respect to binding curve characteristics.

Evidence for the involvement of a carboxyl group in the vicinity of the MK801 and magnesium ion binding site of the N-methyl-D-aspartate receptor.

A series of protein modifying reagents were tested for their effects on the specific binding of [3H]MK801 to adult rat brain membranes. N-Bromosuccinimide, acetyl imidazole, 2,3-butanedione, 5,5'-dithiobis-(2-nitrobenzoic acid) and dithiothreitol all had no significant effect on binding. The carboxylic acid residue modification reagent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC), inhibited [3H]MK801 specific binding in a dose-dependent manner with an IC50 = 1.9 mM. The inhibition by EDAC was due to a decrease in the Bmax with no change in KD. The inhibition of [3H]MK801 binding by EDAC was not prevented by prior incubation with competitive antagonists. Protection against EDAC inactivation was obtained, however, in a dose-dependent manner by preincubation with the divalent cations, Ca2+ and Mg2+, but not Zn2+. These results suggest that EDAC modifies an important carboxyl group located within the voltage-dependent Mg2+ binding site of the N-methyl-D-aspartate receptor. This modification yields a decrease in the specific [3H]MK801 binding activity thus demonstrating a close association between the two allosteric regulatory sites.

Modulation of [3H] TBOB binding to the rodent GABAA receptor by simple disaccharides.

We have recently reported evidence that a simple beta-linked alkylated mannose reversibly increased the magnitude of GABA(A) receptor currents evoked in cultured rat pyramidal neurons whilst concomitantly reducing the incidence of spontaneous synaptic activity. In this present study, the effects of the simple beta-linked disaccharide, lactose was investigated using a [3H] TBOB (t-[3H] butylbicycloorthobenzoate) binding assay in adult rat forebrain and cerebellum membranes. Lactose elicited a significant potentiation of [3H] TBOB binding to well-washed forebrain and cerebellar membranes (mean E(max) values=367 and 287%; mean EC(50) values=1.5 and 30 microM, respectively, N=4). The alpha-linked disaccharides, maltose and sucrose also potentiated [3H] TBOB binding, but with 100-600-fold higher EC(50) values than lactose. The lactose-mediated potentiation of [3H] TBOB in the forebrain and cerebellum was completely abolished in the presence of 0.3 microM GABA. Over the concentration range in which significant potentiation of [3H] TBOB binding was detected, lactose elicited no significant effect upon [3H] flunitrazepam binding. This study demonstrated that lactose can modulate the GABA(A) receptor channel, allosterically coupled to the agonist site, but independent of the benzodiazepine site. Furthermore, lactose displayed differential effects upon forebrain and cerebellar GABA(A) receptors indicating that it may be a novel subtype selective agent.