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Biomedical subjects

I L Martin

Publications and source records attributed to I L Martin.

At least 19 recordsLinked to original sources
PubMed2005

Changes in GABA(A) receptor subunit expression in the midbrain during the oestrous cycle in Wistar rats.

In women, the late luteal phase or "premenstrual" period is commonly associated with psychological disturbances, which include mood changes and increased aggression. The underlying cause is unknown but one possibility is that fluctuations in levels of neuroactive steroids precipitate changes in expression of GABA(A) receptor subunits that result in functional changes in inhibitory control systems. The present study investigated the levels of expression of alpha4, beta1 and delta GABA(A) receptor subunits in the periaqueductal gray matter (PAG) in rats and whether plasticity occurs during the oestrous cycle in females. In male rats alpha4, beta1 and delta subunit immunoreactive neurones were present throughout the PAG in similar numbers. In female rats in proestrus, oestrus and early dioestrus, the density of alpha4, beta1 and delta subunit immunoreactive cells was similar to males. However, in late dioestrus, the numbers increased significantly, especially in the dorsolateral PAG, a region which is particularly rich in GABAergic interneurones. These parallel changes may reflect an increase in expression of the alpha4beta1delta GABA(A) receptor subtype. Recombinant alpha4beta1delta receptors, expressed in Xenopus oocytes, exhibited and EC(50) for GABA an order of magnitude lower (2.02+/-0.33 microM; mean+/-S.E.M.) than that found for the most ubiquitous alpha1beta2gamma2 GABA(A) receptor (32.8+/-2.5 microM). Increased expression of alpha4beta1delta GABA(A) receptors in the interneurones of the PAG could render the panic circuitry abnormally excitable by disinhibiting the ongoing GABAergic inhibition. Similar changes in neuronal excitability within the PAG in women consequent to falling steroid levels in the late luteal phase of the menstrual cycle could contribute to the development of pre-menstrual dysphoria.

Animals↗
PubMed2001

GABA(A) receptor gene expression in rat cortex: differential effects of two chronic diazepam treatment regimes.

Diazepam is widely prescribed as an anxiolytic but its therapeutic application is limited because with daily use tolerance develops to certain aspects of its pharmacological profile. We compared the effects of two dosing paradigms on GABA(A) receptor gene expression and benzodiazepine binding characteristics. Equivalent daily doses of 15 mg/kg/day diazepam were delivered either via constant infusion or daily subcutaneous injection for 14 days. The two distinct treatment regimes produced significantly different changes in GABA(A) receptor alpha4-, beta2-, beta3- and gamma1-subunit mRNA steady-state levels. Similar changes in the GABA enhancement of flunitrazepam binding and the BZ3/BZ2 subtype ratio determined ex vivo were produced, however, significant differences were found in [(3)H]-Ro 15-4513 binding between cortical tissue from diazepam injected animals compared with diazepam infused animals. Our data suggest that it is the diurnal fluctuations in receptor occupancy that are responsible for the different effects produced by these two dosing regimes.

Affinity Labels↗
PubMed2001

Effects of the antidepressant/antipanic drug phenelzine on alanine and alanine transaminase in rat brain.

1. Phenelzine (PLZ) is an antidepressant with anxiolytic properties. Acute and chronic PLZ administration increase brain GABA levels, an effect due, at least in part, to an inhibition of the activity of the GABA metabolizing enzyme, GABA transaminase (GABA-T). 2. Previous preliminary reports have indicated that acute PLZ treatment also elevates brain alanine levels. As with GABA, the metabolism of alanine involves a pyridoxal phosphate-dependent transaminase. 3. In the study reported here, the effects of acute PLZ treatment on the levels of various amino acids, some of which are also metabolized by pyridoxal phosphate-dependent transaminases were compared in rat whole brain. Of the 6 amino acids investigated, only GABA and alanine levels were elevated (in a time- and dose-dependent manner). 4. The elevation in brain alanine levels could be explained, at least in part, by a time- and dose-dependent inhibitory effect of PLZ on alanine transaminase (ALA-T), although as with GABA the increases are higher than expected from the degree of enzyme inhibition produced. In addition, we also showed that the elevation in alanine levels and the inhibition of alanine transaminase in the brain are retained after 14 days of PLZ treatment, and that PLZ produces a marked increase in extracellular levels of alanine. 5. These results are discussed in terms of their relevance to synaptic function and to the pharmacological profile of PLZ.

Alanine↗
PubMed2000

Chiari type I malformation and postoperative respiratory failure.

PURPOSE: To present a case of respiratory failure following suboccipital craniectomy for Chiari type I malformation. CLINICAL FEATURES: A 22-yr-old man presented with a two year history of symptoms and signs suggestive of brainstem compression at the level of the foramen magnum. This was confirmed with magnetic resonance imaging. The procedure of suboccipital craniectomy, upper cervical laminectomy and fourth ventricle exploration was performed. Three hours postoperatively the patient experienced episodes of apnea and subsequently became drowsy. Blood gas analysis revealed hypercapnic respiratory failure. Chest X-ray revealed evidence of pulmonary aspiration. The trachea was re-intubated and the lungs ventilated in intensive care for 72 hr. He was discharged home two weeks postoperatively. CONCLUSION: Chiari type I malformation is associated with a number of associated anomalies. These patients are at considerable risk of respiratory depression and bulbar dysfunction in the perioperative period. The anesthetic issues are reviewed and discussed.

Adult↗
PubMed2000

Intracerebral hemorrhage and Moyamoya disease in pregnancy.

PURPOSE: To present a case of Moyamoya disease with intracranial hemorrhage complicating pregnancy. CLINICAL FEATURES: A 36-yr-old parturient at 34 wk gestation presented with left hemiparesis, headache, nausea and vomiting. Subsequent deterioration in level of consciousness and the development of a dilated right pupil necessitated immediate intubation. Urgent non-contrast CT scan revealed a large right intracerebral hematoma with transtentorial herniation. The patient underwent simultaneous emergency Cesarean section and craniotomy. A postoperative angiogram revealed findings consistent with Moyamoya disease. The neonate survived but the patient developed severe cerebral edema and died eleven days postoperatively. CONCLUSION: Adult patients with Moyamoya disease often present with intracranial hemorrhage which poses unique anesthetic challenges. We report a case of intracerebral hemorrhage during pregnancy, which is known to be associated with high morbidity and mortality. The anesthetic techniques are reviewed and discussed.

Adult↗
PubMed2000

Characterization of the interaction of zopiclone with gamma-aminobutyric acid type A receptors.

Zopiclone is a cyclopyrrolone that is used clinically as a hypnotic. Although this drug is known to interact with neuronal gamma-aminobutyric acid type A receptors, its binding site(s) within the receptor oligomer has been reported to be distinct from that of the classical benzodiazepines. After photoaffinity labeling with flunitrazepam, receptors in rat cerebellar membranes showed differentially reduced affinity for flunitrazepam and zopiclone by 50- and 3-fold, respectively. Because histidine 101 of the alpha-subunit is a major site of photolabeling, we have made specific substitutions of this residue and studied the consequences on the binding properties of zopiclone and diazepam using recombinant alpha1beta2gamma2-receptors transiently expressed in tsA201 cells. Both compounds showed similar binding profiles with receptors containing mutated alpha-subunits, suggesting a similar interaction with the residue at position 101. At alpha1beta2gamma3-receptors, flunitrazepam affinity was dramatically decreased by approximately 36-fold, whereas the affinity for zopiclone was decreased only 3-fold, suggesting a differential contribution of the gamma-subunit to the binding pocket. Additionally, we used electrophysiological techniques to examine the contribution of the gamma-subunit isoform in the receptor oligomer to ligand recognition using recombinant receptors expressed in Xenopus oocytes. Both compounds are agonists at alpha1beta2gamma2- and alpha1beta2gamma3-receptors, with flunitrazepam being more potent but less efficacious. In summary, these data suggest that histidine 101 of the alpha1-subunit plays a similar role in ligand recognition for zopiclone, diazepam, and flunitrazepam.

Amino Acid Substitution↗
PubMed2000

Importance of phenylalanine 107 in agonist recognition by the 5-hydroxytryptamine(3A) receptor.

The 5-hydroxytryptamine (5-HT)(3) receptor is a member of the ligand-gated ion channel receptor family with significant homology to the nicotinic acetylcholine, gamma-aminobutyric acid(A), and glycine receptors. In this receptor class, the agonist binding site is formed by parts of the extracellular amino-terminal region. This study examines the effects of altering phenylalanine 107 (F107) of the 5-HT(3AL) subunit, obtained from NG108-15 cells, using site-directed mutagenesis. The wild-type (WT) and mutant receptors were expressed in HEK 293 cells and characterized using both whole-cell patch-clamp and radioligand binding. The tyrosine mutant F107Y exhibits a significantly lower affinity for the agonist 5-HT (K(i) = 203 versus 15.6 nM) and an increase of similar magnitude in the EC(50) value (10.6 versus 1.2 microM) compared with WT. The activation kinetics of the maximal currents generated by 5-HT with this mutant were markedly slower than those of the WT receptor, but application of supramaximal concentrations of the agonist markedly decreased the time to half-peak. The asparagine mutant F107N displayed a significantly higher affinity for 5-HT than the WT receptor (1.62 versus 15.6 nM), which was mirrored in direction and magnitude by changes in the EC(50) value for this agonist (0.2 versus 1.2 microM). In contrast to the WT receptor, the mutant F107N was activated by acetylcholine (EC(50) = 260 microM). The response to acetylcholine was blocked by the 5-HT(3) receptor antagonist renzapride with a similar IC(50) value as that determined against currents generated by 5-HT in the WT receptor. These data suggest that F107 is an important determinant of agonist recognition at the 5-HT(3) receptor.

Animals↗
PubMed1999

The effect of treatment regimen on the development of tolerance to the sedative and anxiolytic effects of diazepam.

RATIONALE: Chronic treatment with benzodiazepines results in tolerance to their sedative and anxiolytic effects and there is considerable evidence that different mechanisms underlie the development of tolerance to different behavioural effects. OBJECTIVE: The purpose of the present experiment was to compare the behavioural effects of chronic treatment with diazepam (15 mg/kg per day) given as daily subcutaneous injections or by osmotic minipump. Both regimens resulted in continual receptor occupancy, but the daily injections also provided a period of higher brain concentrations. METHODS: Rats were tested in the holeboard, which provides measures of exploration and locomotor activity, and in the elevated plus-maze and social interaction tests of anxiety. For those in the subcutaneous injection group the tests were 2 h after injection, when brain concentrations were highest. RESULTS: Despite a higher brain concentration in the injected group, both groups showed tolerance to diazepam's sedative effects, after 7 days of treatment. In contrast, in the elevated plus-maze, there was tolerance to the anxiolytic effects in the pump group after 14 days, but a persisting anxiolytic effect in the injected group at 14 and 28 days. Whilst higher brain concentrations could explain this result in the plus-maze, they cannot account for the pattern observed in the social interaction test, where the injection group showed a significant anxiogenic effect at 28 days. CONCLUSIONS: Whereas the mechanism underlying tolerance to the sedative effects of diazepam was insensitive to the different treatment regimens, the results suggest that different adaptive mechanisms were triggered in the two tests of anxiety with a differential sensitivity to the treatment regimen. The adaptive mechanism predominating in the social interaction test was favoured by the injection regimen which produced intermittent peak concentrations. This mechanism seems to be an oppositional one, leading to an anxiogenic response, which was manifest despite high brain concentrations of diazepam at the time of testing.

Animals↗
PubMed1999

Decreased GABA enhancement of benzodiazepine binding after a single dose of diazepam.

The GABA and benzodiazepine binding sites on GABA(A) receptors are allosterically coupled. The in vitro binding of 2 nM [3H]flunitrazepam to cortical and cerebellar membranes prepared from drug-naive rats was potentiated approximately 1.6-fold by 100 microM GABA. Potentiation in both regions was significantly reduced 4 or 12 but not 24 h after a single dose of 15 mg/kg diazepam. At 24 h after the last of 14 daily doses of diazepam, no differences in GABA potentiation were observed. Diazepam-induced changes in GABA(A) receptor gamma2-subunit gene transcription and alpha1-, beta2-, and gamma2-subunit steady-state mRNA levels did not appear to be temporally related to allosteric uncoupling.

Animals↗
PubMed1997

Rapid, sensitive procedure to determine buspirone levels in rat brains using gas chromatography with nitrogen-phosphorus detection.

Reported here is a rapid, sensitive and relatively inexpensive procedure using gas chromatography with nitrogen-phosphorus detection (GC-NPD) to quantify buspirone levels in brains of rats. The analyte was directly extracted from brain homogenate with toluene after basification and then subjected to GC-NPD analysis using a capillary column. The calibration curves were linear over the range of 10 to 320 ng per 2 ml of brain homogenate, with typical r2 values >0.99. The assay was highly reproducible and gave peaks with excellent chromatographic properties.

Animals↗
PubMed1997

Chronic zolpidem treatment alters GABA(A) receptor mRNA levels in the rat cortex.

The effect of chronic zolpidem treatment on the steady-state levels of gamma-aminobutyric acidA alpha1-6, beta1-3 and gamma1-3 subunit mRNAs in rat cortex has been investigated. Male Sprague-Dawley rats were injected once daily, for 7 or 14 days, with 15 mg/kg of zolpidem in sesame oil vehicle. The levels of the alpha4 and beta1 subunit mRNAs were significantly increased after 7 days of treatment and the level of alpha1 subunit mRNA was significantly decreased after 14 days of treatment, as determined by solution hybridization. These results are compared to the previously determined effects of an equivalent schedule of treatment with diazepam.

Animals↗
PubMed1997

Chronic diazepam exposure decreases transcription of the rat GABA(A) receptor gamma2-subunit gene.

The rate of transcription of the GABA(A) receptor gamma2-subunit gene in rat cortex has been measured using the nuclear run-off transcriptional assay. Exposure of rats to diazepam (15 mg/kg/day for 14 days) caused a significant reduction in the level of nascent GABA(A) receptor gamma2-subunit transcripts. Therefore, a component of the cellular response to chronic benzodiazepine exposure includes events which take place at the level of transcription of a GABA(A) receptor gene.

Animals↗
PubMed1996

The major site of photoaffinity labeling of the gamma-aminobutyric acid type A receptor by [3H]flunitrazepam is histidine 102 of the alpha subunit.

The alpha subunit of the gamma-aminobutyric acid type A (GABA(A)) receptor is known to be photoaffinity labeled by the classical benzodiazepine agonist, [3H]flunitrazepam. To identify the specific site for [3H]flunitrazepam photoincorporation in the receptor subunit, we have subjected photoaffinity labeled GABA(A) receptors from bovine cerebral cortex to specific cleavage with cyanogen bromide and purified the resulting photolabeled peptides by immunoprecipitation with an anti-flunitrazepam polyclonal serum. A major photolabeled peptide component from reversed-phase high performance liquid chromatography of the immunopurified peptides was resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The radioactivity profile indicated that the [3H]flunitrazepam photoaffinity label is covalently associated with a 5.4-kDa peptide. This peptide is glycosylated because treatment with the enzyme, peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, reduced the molecular mass of the peptide to 3.2 kDa. Direct sequencing of the photolabeled peptide by automated Edman degradation showed that the radioactivity is released in the twelfth cycle. Based on the molecular mass of the peptides that can be generated by cyanogen bromide cleavage of the GABA(A) receptor alpha subunit and the potential sites for asparagine-linked glycosylation, the pattern of release of radioactivity during Edman degradation of the photolabeled peptide was mapped to the known amino acid sequence of the receptor subunit. The major site of photoincorporation by [3H]flunitrazepam on the GABA(A) receptor is shown to be alpha subunit residue His102 (numbering based on bovine alpha 1 sequence).

Affinity Labels↗
PubMed1996

Dimethyl sulfoxide/propylene glycol is a suitable solvent for the delivery of diazepam from osmotic minipumps.

The functional integrity of Alzet osmotic minipumps was assessed using two organic solvents (50% (v/v) dimethyl sulfoxide (DMSO)/50% (v/v) propylene glycol (PG) and 100% tetraglycol) which dissolve diazepam, an aqueous insoluble benzodiazepine. Both solvents showed a significant decrease in output rate over time: the decline with tetraglycol was, however, more marked and variable. Further, the DMSO/PG vehicle demonstrated a comparable decline in rate (1.45%) to that of the control vehicles saline and water (1.12%). DMSO/PG is therefore a suitable solvent for the chronic delivery of diazepam from osmotic minipumps.

Animals↗
PubMed1996

Chronic treatment with diazepam or abecarnil differently affects the expression of GABAA receptor subunit mRNAs in the rat cortex.

Diazepam and abecarnil produce their overt effects by interaction with the GABAA receptor. Chronic treatment with abecarnil, however, does not induce diazepam-like tolerance. This study investigates the effects of chronic diazepam and abecarnil treatment on expression of GABAA receptor alpha 1-6 beta 1-3 and gamma 1-3 subunit isoform mRNAs in rat cortex. Male Sprague-Dawley rats were injected subcutaneously once daily for 7 or 14 days with 15 mg/kg diazepam or 6 mg/kg abecarnil in sesame-oil vehicle, and steady-state levels of GABAA receptor subunit mRNAs were quantified by solution hybridization. The levels of alpha 4- and alpha-, beta 1- and gamma 3-subunit mRNAs were significantly increased after 7 days of diazepam treatment, and this effect was maintained at 14 days. A significant increase in alpha 3-subunit mRNA was apparent only after 14 days of diazepam treatment and a significant decrease in beta 2-subunit mRNA was seen only after 14 days of abecarnil treatment. Gamma 2-Subunit mRNA was significantly decreased after 14 days of either diazepam or abecarnil exposure. A degree of association between a particular drug treatment and changes in the levels of mRNAs arising from a given gene cluster was noted. Our results are consistent with a model of diazepam dependence based on GABAA receptor subunit isoform switching.

Animals↗
PubMed1996

Identification of domains in human recombinant GABAA receptors that are photoaffinity labelled by [3H]flunitrazepam and [3H]Ro15-4513.

We have used [3H]flunitrazepam and [3H]Ro15-4513 as photoaffinity labelling agents in combination with a chemical cleavage technique to localize the benzodiazepine recognition sites of specific human recombinant alpha 1 beta 1 gamma 2, alpha 1 beta 3 gamma 2 and alpha 6 beta 3 gamma 2 GABAA receptor subtypes. The chemical agent utilized was hydroxylamine, whose substrate is a relatively rare asparagine-glycine amide bond that occurs only in the alpha subunits of the receptors examined in this study. Cleavage products were resolved using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results of these experiments show that, in the alpha 1 subunit-containing receptors, incorporation of [3H]flunitrazepam occurs within residues 1-103 of the alpha 1 subunit, while incorporation of [3H]Ro15-4513 occurs within the region of the alpha 1 subunit that lies between residue 104 and the C-terminus. Photolabelling of membranes prepared from the alpha 6 beta 3 gamma 2-expressing cell line with [3H]Ro15-4513 resulted in the incorporation of radiolabel into two major protein species of M(r) 56,000 and M(r) 48,000, indicating incorporation into the alpha 6 subunit and possibly also the gamma 2 subunit. Hydroxylamine cleavage of alpha 6-containing receptors labelled with [3H]Ro15-4513 produced a gel profile consistent with the incorporation of the label occurring between residue 125 and the C-terminal. Thus, we have shown that the recognition sites for the agonist [3H]flunitrazepam and the inverse agonist [3H]Ro15-4513 occur within distinct domains of the human GABAA receptor.

Affinity Labels↗
PubMed1995

Ultrastructure of the 5-hydroxytryptamine3 receptor.

We have determined the ultrastructure of 5-hydroxytryptamine3 (5-HT3) serotonin receptors purified from NG108-15 mouse neuroblastoma x rat glioma cells by electron microscopic examination of receptor particles embedded in uranyl acetate stain and metal replicas of rapidly frozen receptors. The 5-HT3 receptor can be modelled as a cylinder 11 nm in length and 8 nm in diameter with a closed end and a central cavity 3 nm in diameter. Analysis of the rotational symmetry of single receptor particles indicates that the 5-HT3 receptor is composed of five subunits arranged symmetrically around a central cavity. Together with evidence obtained for related proteins in other studies using ultrastructural, biochemical, or electrophysiological methods, our observations suggest that all members of the ligand-gated ion channel superfamily may possess a pentameric quaternary structure.

Animals↗