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M B Robinson

Publications and source records attributed to M B Robinson.

At least 73 records · Page 4Linked to original sources
PubMed1995

The sparse fur mouse as a model for gene therapy in ornithine carbamoyltransferase deficiency.

The sparse fur (spf/Y) mouse was evaluated as a model for studying gene therapy in ornithine carbamoyltransferase deficiency (OCTD), the most common inborn error of urea synthesis. Previous studies have defined a number of biochemical characteristics of this animal model that are analogous to the human disease: OCTD in liver, elevated ammonium and glutamine, low citrulline and arginine in plasma, elevated urinary orotic acid excretion, neurochemical alterations and responsiveness to alternative pathway therapy. In this study, metabolic flux, survival, behavior and learning of these animals were examined in preparation for a trial of gene therapy. We found that, as has been previously reported, OCT activity in liver ranged from 10 to 20% of control. Yet, stable isotope studies using 15N ammonium chloride to follow ureagenesis in vivo showed 55% of normal urea synthetic capacity. This suggests that partial correction with gene therapy may be sufficient to normalize urea synthesis. Although it has been suggested that liver OCTD and its consequent metabolic effects normalize without treatment by adulthood in the spf/Y mouse, we did not find this to be the case. We documented that the spf/Y mouse had a markedly decreased lifespan (< 10% of normal) and remained runted throughout life. In terms of behavior, the spf/Y mice had evidence of decreased learning in a passive avoidance task that was not attributable to alterations in activity. These clearly definable metabolic and behavioral abnormalities suggest that the spf/Y mouse should prove a useful model for studying the efficacy of gene therapy in OCTD.

Amino Acid Metabolism, Inborn Errors↗
PubMed1994

The effects of L-glutamate and trans-(+-)-1-amino-1,3-cyclopentanedicarboxylate on phosphoinositide hydrolysis can be pharmacologically differentiated.

The excitatory amino acid analogues L-glutamate (L-Glu), L-aspartate (L-Asp), D-Asp, and trans-(+-)-1-amino-1,3-cyclopentanedicarboxylate (trans-ACPD) stimulate the hydrolysis of phosphoinositides (PI). In the present studies, the effects of noncompetitive and competitive inhibitors on PI hydrolysis stimulated by excitatory amino acid analogues were examined. When agonist and inhibitor were added simultaneously to hippocampal tissue, the noncompetitive inhibitor L-2-amino-3-phosphonopropionate (L-AP3) did not block the effects of L-Glu, L-Asp, or D-Asp at concentrations that block the effects of trans-ACPD by more than 80%. When tissue was preincubated with L-AP3, the effects of L-Glu, L-Asp, or D-Asp were blocked (IC50 values between 65 and 210 microM). Unlike L-AP3, L-aspartate-beta-hydroxamate (L-A beta HA) inhibited PI hydrolysis stimulated by trans-ACPD, L-Glu, L-Asp, or D-Asp when agonist and inhibitor were added simultaneously in hippocampus; its effects were not time-dependent. In cerebellum, both L-AP3 and L-A beta HA had agonist activity. Inhibition by the recently identified competitive inhibitor (+)-alpha-methyl-4-carboxyphenylglycine [(+)-MCPG] of PI hydrolysis was also examined. (+)-MCPG blocked PI hydrolysis stimulated by trans-ACPD, L-Asp, or D-Asp in both hippocampus and cerebellum (IC50 values between 220 and 1,700 microM). The effects of (+)-MCPG were consistent with a competitive mechanism of action. (+)-MCPG (up to 3 mM) blocked PI hydrolysis stimulated by L-Glu by less than 25% in both hippocampus and cerebellum.

Alanine↗
PubMed1994

Evaluation of medical audit.

OBJECTIVE: To review current knowledge of the effectiveness of medical audit programmes as a whole and of specific interventions within these programmes, as a means of changing clinical behaviour. CRITERIA FOR INCLUSION AND EXCLUSION OF PUBLISHED REPORTS: Articles listed on Medline from 1985-92 with key words "quality assurance" or "medical audit", and "evaluation" and relevant references from these articles, and from recently published reviews and reports on medical audit, were included. Excluded were simple descriptions of audit activity, replications of previous work, and publication in a language other than English. RESULTS: Evaluation of entire programmes of medical audit is unusual. Most reports concern specific interventions and focus particularly on the scientific and technical aspects of quality. These interventions may be classified by the means through which they attempt to achieve desired changes: patient characteristics; physician characteristics; administrative and organisational structures; and financial incentives. CONCLUSIONS: Knowledge about effective methods of bringing about specific changes in clinical behaviour is rudimentary. Impact is highly dependent on local factors, so generalisation of results to other settings is difficult. More qualitative research is needed to define the local factors which influence results.

Attitude of Health Personnel↗
PubMed1994

An animal model of early-treated PKU.

Phenylketonuria (PKU) is a genetic disorder in which the hydroxylation of phenylalanine (Phe) to tyrosine is severely disrupted. If PKU is left untreated, severe mental retardation results. The accepted treatment is to restrict dietary intake of Phe. It has generally been thought that cognitive impairments are prevented if levels of Phe in plasma are maintained at or below five times the normal level. However, we recently documented that children treated early and continuously for PKU or children mildly hyperphenylalaninemic, who have levels of Phe in plasma approximately three to five times normal, still have cognitive impairments. These impairments are specific to the functions of frontal cortex (A. Diamond, W. Hurwitz, E. Lee, W. Grover, and C. Minarcik, unpublished observations). To investigate the mechanism underlying these cognitive deficits, an animal model of this condition was developed and characterized. Thirty-six rat pups were divided into three groups. The first group was treated pre- and postnatally with Phe and alpha-methylphenylalanine (a phenylalanine hydroxylase inhibitor). The second group was injected postnatally with Phe and alpha-methylphenylalanine. The third group received postnatal control injections. The mild plasma Phe elevations in the two experimental groups produced significant behavioral and neurochemical effects. Both experimental groups were impaired on a task dependent on frontal cortex, delayed alternation. Levels of dopamine, homovanillic acid (HVA), norepinephrine, and 5-hydroxyindole acetic acid (5-HIAA) were measured in medial prefrontal cortex, anterior cingulate cortex, striatum, and nucleus accumbens. The largest neurochemical reductions observed were in HVA and were in the two frontal cortical areas (medial prefrontal cortex and anterior cingulate cortex). There were modest reductions in HVA in the nucleus accumbens but no significant changes in HVA, or in any other metabolite or neurotransmitter, in the striatum. The levels of 5-HIAA were also reduced in all brain regions examined. There was no effect on norepinephrine in any of the four regions examined. Reduced levels of HVA in medial prefrontal cortex were the only neurochemical effect that significantly correlated with every measure of performance on the delayed alternation task. This study provides evidence of deleterious effects from mild elevations in the levels of Phe in plasma previously considered small enough to be safe. These effects include impaired performance on a cognitive task dependent on frontal cortex and reduced HVA levels in frontal cortex.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

The glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate depresses excitatory synaptic transmission via a presynaptic mechanism in cultured hippocampal neurons.

Sodium-dependent high-affinity uptake of glutamate is thought to play a major role in the maintenance of very low extracellular concentrations of excitatory amino acids (EAA), and may modulate the actions of released transmitter at G-protein-coupled receptors and extrasynaptic receptors that are activated over a longer distance and time course. We have examined the effects of the recently developed uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (L-trans-PDC) on monosynaptically evoked excitatory postsynaptic currents (EPSCs) in very-low-density cultures of hippocampal neurons. L-Trans-PDC produced a decreased amplitude of both the non-NMDA and NMDA receptor-mediated components of monosynaptically evoked EPSCs. Examination of miniature EPSCs (mEPSCs) indicated that changes in the sensitivity of postsynaptic non-NMDA receptors did not underline the decrease in evoked EPSC amplitudes. The metabotropic receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) also depressed both components of the EPSC. The competitive metabotropic receptor antagonist (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG) blocked the depression of EPSC amplitude induced by 1S,3R-ACPD and also blocked the effects of L-trans-PDC. Finally, low concentrations of L-glutamate (2 microM) mimicked the effects of L-trans-PDC on EPSC amplitude. From these results we conclude that the application of L-trans-PDC to cultured hippocampal neurons results in the activation of presynaptic metabotropic receptors, leading to a decrease in synaptic transmission. We propose that this effect is due to an increase in ambient glutamate concentrations following inhibition of glutamate uptake, resulting in presynaptic inhibition of excitatory synaptic transmission.

Animals↗
PubMed1993

Repeated exposure to hyperbaric oxygen sensitizes rats to oxygen-induced seizures.

Repeated exposure to increased partial pressure of oxygen (PO2) is the standard of care for several medical conditions. The side-effects of repeated exposure to hyperbaric oxygen (HBO), however, are not well defined. Previous studies have demonstrated that acute exposure of rats to HBO causes hypothermia that precedes convulsions. In the present studies, rats that were repeatedly exposed to 100% oxygen at 4 atmospheres absolute (ATA) pressure developed convulsions earlier than naive controls. There was also a trend toward less hypothermia in the rats repeatedly exposed to oxygen. The purpose of this study was to test the hypothesis that repeated exposure to HBO increases sensitivity to convulsions induced by HBO and to determine if the time to onset of convulsions is affected by the hypothermia caused by exposure to HBO. Rats were repeatedly exposed to 2 ATA oxygen for a total of 10 days. After 72 h, these rats were challenged by exposure to 100% oxygen at 4 ATA pressure. Rats repeatedly exposed to HBO had convulsions significantly earlier than the naive controls (84 +/- 8 min compared to 147 +/- 11 min), and they developed significantly less hypothermia. Control studies suggested that the decrease in the degree of hypothermia was caused by both repeated exposure to oxygen and adaptation to the mild restraint used during oxygen re-exposures. Adaptation to restraint eliminated the hypothermia induced by oxygen but did not change the time to onset of convulsions. Increased sensitivity to convulsions was present after five exposures to 2 ATA oxygen and persisted for 10 days after the last 2 ATA oxygen re-exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1993

Quinolinic acid in children with congenital hyperammonemia.

Levels of the excitotoxin quinolinic acid (QUIN) were measured in the cerebrospinal fluid of infants and children with congenital hyperammonemia. Twofold to tenfold elevations of QUIN were found in 4 neonates in hyperammonemic coma (QUIN range, 250-990 nM; control mean, 110 +/- 90 nM; p < 0.005). Similar elevations of neopterin were found (range, 24-75 nM; control mean, 9.0 +/- 4.9 nM; p < 0.005). In addition, significant elevations of QUIN were found in 14 older children with congenital hyperammonemia (mean, 50 +/- 20 vs 17 +/- 6 nM; p < 0.05). Neopterin levels were not elevated in these children. The QUIN may originate from an increase in tryptophan transport across the blood-brain barrier or from induction of indolamine-2,3-dioxygenase activity. These findings support a role for QUIN in the neuropathology of congenital hyperammonemia. They also suggest the potential utility of N-methyl-D-aspartate receptor-blocking agents or inhibitors of QUIN synthesis in the treatment of hyperammonemic coma.

Ammonia↗
PubMed1993

Hypothermia induced by hyperbaric oxygen is not blocked by serotonin antagonists.

Exposure to HBO causes hypothermia, bradycardia, head weaving, resting tremor, piloerection, and straub tail in rats. These physiological and behavioral responses can also be evoked by selective activation of serotonin1A (5-HT1A) receptors. The purpose of the current study was to determine if hypothermia caused by HBO is due to increased activation of 5-HT1A receptors. The levels of brain biogenic amines were measured in brain regions of Sprague-Dawley (SD) rats exposed to HBO. Exposure to HBO caused an increase in the levels of 5-hydroxyindoleacetic acid (5-HIAA) in the striatum (92%, p < 0.05) and occipital-temporal cortex (116%, p < 0.05), but not in other brain regions. Exposure to HBO did not change the levels of tryptophan, serotonin (5-HT), other biogenic amines, or their metabolites. It is hypothesized that the Fawn Hood (FH) rat, which is reported to be resistant to hypothermia induced by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), has an abnormality of 5-HT1A receptor activity. Although the FH rat was more resistant to hypothermia induced by HBO than the SD rat, we were not able to confirm that this rat was resistant to hypothermia induced by 8-OH-DPAT. The 5-HT receptor antagonists, 1-(1H-Indol-4-yloxy)-3-[(1-methylethyl)amino]-2-propanol (Pindolol), 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl] piperazine hydrobromide (NAN-190), and methysergide, did not block hypothermia induced by HBO in SD rats. A series of control experiments were used to confirm that the antagonists blocked hypothermia induced by serotonin agonists.(ABSTRACT TRUNCATED AT 250 WORDS)

5-Hydroxytryptophan↗
PubMed1993

Multiple subtypes of excitatory amino acid receptors coupled to the hydrolysis of phosphoinositides in rat brain.

The excitatory amino acid (EAA) analogues quisqualate, ibotenate, and trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylate (trans-ACPD) activate the metabotropic EAA receptors that are coupled to the hydrolysis of phosphoinositides (PI). Previous studies of hippocampal cross sections demonstrated that PI hydrolysis stimulated by these agonists can be inhibited by either L-aspartate-beta-hydroxamate (L-A beta HA) or DL-2-amino-3-phosphonopropionate (DL-AP3). The goal of the present studies was to determine if all metabotropic EAA receptors are sensitive to L-A beta HA and DL-AP3. Two approaches were used. In the first, using cerebellar cross sections, the effects of these agonists and inhibitors were examined. The EC50 values (the concentrations required to evoke half-maximal stimulation) of quisqualate, ibotenate, and trans-ACPD in cerebellum were similar to the EC50 values that we observed previously in hippocampus, but neither L-A beta HA nor DL-AP3 blocked PI hydrolysis. The EC50 values were 0.65 +/- 0.17 microM for quisqualate, 12.8 +/- 2.5 microM for ibotenate, and 18.1 +/- 3.1 microM for trans-ACPD. All data were best fit to theoretical curves that had Hill slopes of 1. In the second approach, another EAA analogue, D-aspartate, was identified as an agonist that stimulates PI hydrolysis. The EC50 for PI hydrolysis stimulated by D-aspartate was 470 +/- 90 microM in hippocampus. Neither L-A beta HA nor DL-AP3 blocked PI hydrolysis stimulated by D-aspartate in hippocampus.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Subtypes of sodium-dependent high-affinity L-[3H]glutamate transport activity: pharmacologic specificity and regulation by sodium and potassium.

Some data suggest that the sodium-dependent, high-affinity L-glutamate (Glu) transport sites in forebrain are different from those in cerebellum. In the present study, sodium-dependent transport of L-[3H]Glu was characterized in cerebellum and cortex. In both cerebellar and cortical tissue, activity was enriched in synaptosomes. Approximately 100 excitatory amino acid analogues were tested as potential inhibitors of transport activity. Many of the compounds tested inhibited transport activity by < 65% at 1 mM and were not studied further. One group of compounds exhibited inhibition conforming to theoretical curves with Hill coefficients of 1 and were < 10-fold selective as inhibitors of transport activity. These included three of the putative endogenous substrates for transport: L-Glu, L-aspartate, and L-cysteate. Four of the compounds exhibited inhibition conforming to theoretical curves with Hill coefficients of 1 and were > 10-fold selective as inhibitors. These included beta-N-oxalyl-L-alpha,beta-diaminopropionate, alpha-methyl-DL-glutamate, (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine, and (2S,1'S,2'S,3'S)-2-(2-carboxy-3-methoxymethylcyclopropyl)glycine. Data obtained with a few of the inhibitors were consistent with two sites in one or both of the brain regions. (2S,1'R,2'R)-2-(Carboxycyclopropyl)glycine (L-CCG-II) was identified as the most potent (IC50 = 5.5 microM) and selective (60-100-fold) inhibitor of transport activity in cerebellum. One of the potential endogenous substrates, L-homocysteate, was also a selective inhibitor of cerebellar transport activity. The data for inhibition of transport activity in cortex by both L-CCG-II and L-homocysteate were best fit to two sites. Kainate was equipotent as an inhibitor of transport activity, and in both brain regions the data for inhibition were best fit to two sites. The possibility that there are four subtypes of excitatory amino acid transport is discussed. Altering sodium and potassium levels affects cerebellar and cortical transport activity differently, suggesting that the differences extend to other recognition sites on these transporters.

Analysis of Variance↗
PubMed1993

Inhibition of glutamate uptake with L-trans-pyrrolidine-2,4-dicarboxylate potentiates glutamate toxicity in primary hippocampal cultures.

Sodium-dependent, high-affinity glutamate transport is generally assumed to limit the toxicity of glutamate in vivo and in vitro, but there is very little direct evidence to support this hypothesis. In the present study, the effects of the specific uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate on the toxicity and clearance of glutamate were examined in hippocampal neuronal cultures. At a concentration that was not toxic by itself, L-trans-pyrrolidine-2,4-dicarboxylate increased the toxicity of glutamate approximately fivefold and slowed the clearance of glutamate from the extracellular space. This toxicity was almost completely blocked by the N-methyl-D-aspartate receptor antagonist, D-2-amino-5-phosphonopentanoate. These studies provide direct evidence that sodium-dependent, high-affinity glutamate transport limits glutamate toxicity in vitro.

Animals↗
PubMed1993

Which maternity unit? Who makes the choice?

To investigate the factors determining the choice of maternity unit in an outer London district where half of all births occur outside the district, a self-administered questionnaire was distributed by community midwives at postnatal home visits. The main outcome measures were: (1) proportion of mothers able to deliver in unit of first choice; (2) proportion of mothers making a personal choice rather than taking advice from their GP; (3) reasons for not choosing district unit, by parity. It was found that 28/166 (17 per cent) of mothers were unable to deliver in their first choice of unit, and 3/166 (2 per cent) were unable to obtain their second choice. Of the women who did obtain their first choice, 84/136 (62 per cent) had made a personal decision rather than being advised or told by their GP; 58 of these 84 personal deciders went outside the district, but for 36/58 (62 per cent) this was either because the other unit was nearer their home or because they had delivered there before. It is concluded that, although GPs are theoretically free to refer to any maternity unit, a sizeable minority of women are unable to realize their own choices. GPs seem to regulate flows by advising women without strong personal views to attend the local unit. Differences in the quality of care as perceived either by women or their GPs do not seem particularly important in determining choices. The introduction of an internal market in maternity care seems unlikely to result in improved quality of care.

Adult↗
PubMed1992

Brain serotonin2 and serotonin1A receptors are altered in the congenitally hyperammonemic sparse fur mouse.

In previous studies we documented an increase in the levels of the serotonin metabolite, 5-hydroxyindoleacetic acid, in the congenitally hyperammonemic sparse fur mouse. To extend these findings, brain serotonin receptors were studied in these animals. Radioligand binding assays were performed using [3H]ketanserin to label serotonin2 sites and 8-[3H]hydroxy(di-n-propylamino)tetralin to label serotonin1A sites in cortical membrane homogenates. The capacity (Bmax) for [3H]ketanserin binding was significantly lower (-21%; p less than 0.05) in sparse fur animals than in control animals; there was no change in affinity (KD). In contrast, the capacity for 8-[3H]hydroxy(di-n-propylamino)tetralin binding was significantly greater (26%; p less than 0.05) in sparse fur compared with control animals. No difference in affinity was observed. Using two behavioral assays, the functional responsiveness of these serotonin receptors was compared in sparse fur and control animals. Head twitch activity elicited by administration of the serotonin agonist quipazine was studied as a behavior mediated by serotonin2 receptors. Compared with controls, sparse fur mice demonstrated a significantly decreased head twitch response (p less than 0.005). Hypothermia elicited by administration of 8-hydroxy(di-n-propylamino)tetralin was studied as a physiologic response mediated by serotonin1A receptors. Although there were not overall group differences in the dose-response data, there was a significant increase in the hypothermia induced by 8-hydroxy(di-n-propylamino)tetralin in sparse fur compared with control mice (p less than 0.02) at the highest dose. These data provide further support for a link between hyperammonemia and alterations in the serotonin system.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1992

Multiple mechanisms for inhibition of excitatory amino acid receptors coupled to phosphoinositide hydrolysis.

Excitatory amino acid (EAA) analogues activate receptors that are coupled to the increased hydrolysis of phosphoinositides (PIs). In these studies, hippocampal slices were prepared from neonatal rats (6-11 days old) to characterize the effects of EAA analogues on these receptors. The concentrations of ibotenate and trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylate (trans-ACPD) required to evoke half-maximal stimulation (EC50 values) were 28 and 51 microM, respectively. Although the data for stimulation of PI hydrolysis by ibotenate and trans-ACPD were best fit to theoretical curves that had Hill slopes of 1, data for stimulation of PI hydrolysis by quisqualate were best fit to two sites. The EC50 values were 0.43 and 44 microM. The high-affinity sites were 70% of the total. A number of EAA analogues were tested for inhibition of PI metabolism. One of these, L-aspartate-beta-hydroxamate (L-A beta HA), was identified as a novel inhibitor of this response. L-A beta HA was equipotent as an inhibitor of PI metabolism stimulated by ibotenate, quisqualate, and trans-ACPD. The data for this inhibition were best fit to two sites. Between 32 and 48% of the total sites had high affinity with IC50 values in the range of 1.2-6.3 microM. The low-affinity sites had IC50 values between 610 and 2,700 microM. DL-2-Amino-3-phosphonopropionate (DL-AP3) was also equipotent as an inhibitor of PI hydrolysis stimulated by ibotenate, quisqualate, and trans-ACPD (IC50 values were 480-850 microM). In contrast to the data for L-A beta HA, the data for DL-AP3 were best fit to a single site. Both of these inhibitors reduced the maximal response caused by the agonists, consistent with noncompetitive mechanisms of action. Several experiments were designed to examine potential mechanisms for these noncompetitive effects. These studies suggest that either L-A beta HA and DL-AP3 bind to a site on the receptor and irreversibly block activation of the receptor, or that these inhibitors act via a distinct site that specifically regulates EAA receptors coupled to PI hydrolysis.

Alanine↗
PubMed1992

Quinolinate in brain and cerebrospinal fluid in rat models of congenital hyperammonemia.

Children with inborn errors of urea synthesis who survive neonatal hyperammonemic coma commonly exhibit cognitive deficits and neurologic abnormalities. Yet, there is evidence that ammonia is not the only neurotoxin. Hyperammonemia appears to induce a number of neurochemical alterations. In rodent models of hyperammonemia, uptake of L-tryptophan into brain is increased. It has been reported that in an experimental rat model of hepatic encephalopathy, in the ammonium acetate-injected rat, and in patients with hepatic failure and inborn errors of ammonia metabolism, quinolinate, a tryptophan metabolite, is increased. Elevations in quinolinate are of particular concern, as quinolinate could excessively activate the N-methyl-D-aspartate subclass of excitatory amino acid receptors, thereby causing selective neuronal necrosis. We sought to identify an animal model that would replicate the increases in quinolinate that have been associated with hyperammonemia in humans. Levels of quinolinate were measured in hyperammonemic urease-infused rats and ammonium acetate-injected rats. In the urease-infused rat, brain tryptophan was doubled, and serotonin and its metabolite 5-hydroxyindoleacetic acid were significantly increased. Yet, despite the increase in tryptophan and evidence for increased metabolism of tryptophan to serotonin, there were no observed increases of quinolinate in brain, cerebrospinal fluid, or plasma. In the ammonium acetate-injected rat, significant increases of 5-hydroxyindoleacetic acid in cerebral cortex were also observed, but quinolinate did not change in cerebrospinal fluid or cerebral cortex. In summary, we were unable to demonstrate an increase of quinolinate in brain or cerebrospinal fluid in these rat models of hyperammonemia.

Acetates↗
PubMed1991

Pharmacologically distinct sodium-dependent L-[3H]glutamate transport processes in rat brain.

The transport of L-[3H]glutamate into crude synaptosomal membrane fractions prepared from cerebellum, brainstem, hippocampus, cortex, striatum, and midbrain was characterized. In all brain regions, greater than 95% of the accumulation of radiolabel was sodium-dependent and the concentration-dependence was consistent with a single high affinity site. Dihydrokainate and L-alpha-aminoadipate were region specific inhibitors of uptake; this inhibition was consistent with a competitive mechanism. In the forebrain regions examined, dihydrokainate inhibited transport with IC50s of approx. 100 microM (range from 80 to 170 microM). Transport in cerebellum was essentially dihydrokainate-insensitive L-alpha-Aminoadipate inhibited transport in forebrain regions with IC50s of approx. 700 microM (range from 590 to 800 microM) and inhibited transport in cerebellum with an IC50 of 40 microM. The inhibition data obtained with forebrain and cerebellar tissues were consistent with nearly homogeneous (greater than 80%) populations of non-interacting sites. Inhibition data obtained with tissue prepared from brainstem were best fit to a mixture of the two sites (35-50% of the type observed in cerebellum). Other previously identified uptake inhibitors, including DL-threo-hydroxyaspartate, L-aspartate-beta-hydroxamate, beta-glutamate, and L-cysteine sulfinate were not selective for the two types of transport. These data demonstrate that there are two pharmacologically distinct sodium-dependent high affinity transport systems with heterogeneous regional distributions.

Amino Acids↗
PubMed1990

Rat brain N-acetylated alpha-linked acidic dipeptidase activity. Purification and immunologic characterization.

N-Acetylated alpha-linked acidic dipeptidase (NAALA dipeptidase) is a membrane-bound metallopeptidase that cleaves glutamate from the endogenous neuropeptide N-acetyl-L-aspartyl-L-glutamate. In this report, we have solubilized NAALA dipeptidase activity from synaptosomal membranes with Triton X-100 and purified it to apparent homogeneity by sequential column chromatography on DEAE-Sepharose, CM-Sepharose, and lentil lectin-Sepharose. This procedure resulted in a 720-fold purification with 1.6% yield. The purified ezyme migrated as a single silver-stained band on a sodium dodecyl sulfate gel with an apparent molecular weight of 94 kDa. Using an enzymatic stain to visualize NAALA dipeptidase activity within a gel matrix, we have confirmed that the 94-kDa band is, indeed, NAALA dipeptidase. The purified enzyme was characterized and found to be pharmacologically similar to NAALA dipeptidase activity described previously in synaptosomal membrane extracts. Using the purified NAALA dipeptidase as antigen, we have raised specific and high titer polyclonal antibodies in guinea pig. Immunocytochemical studies show intense NAALA dipeptidase immunoreactivity in the cerebellar and renal cortices.

Animals↗