David R. Bristow

Light microscopic autoradiographic localisation of [3H] glycine and [3H] strychnine binding sites in rat brain

Receptor autoradiography has been employed to determine the distribution of strychnine-insensitive glycine binding sites in rat brain using [3H]glycine as a ligand. The location was significantly different from and more widespread than glycine sensitive [3H]strychnine binding sites. Highest binding densities were observed in hippocampus, cortex, subiculum and amygdala followed by striatum, cerebellum and olfactory areas. Characterisation of the binding indicated that it was saturable, of high affinity, stereoselective and displaced by structurally related amino acids. The results support the existence of two glycine receptor subtypes: strychnine-sensitive and strychnine-insensitive.

Effects of tachykinins on inositol phospholipid hydrolysis in slices of hamster urinary bladder

1 Tachykinin‐stimulated inositol phospholipid hydrolysis was examined in slices of hamster urinary bladder. 2 In the presence of lithium, to inhibit inositol monophosphatase activity, substance P, eledoisin and related tachykinins induced large, dose‐dependent increases in [3H]‐inositol monophosphate accumulation. 3 The responses to substance P and eledoisin were not antagonized by the cholinoceptor antagonist, atropine. 4 The rank order of potency for various tachykinins was kassinin ≫ neurokinin A ≫ neurokinin B ≫ eledoisin ≫ physaelamin ≫ substance P ≫ substance P methyl ester. 5 The synthetic analogue [p‐Glu6, D‐Pro9] SP (6–11) was considerably more potent than its L‐prolyl stereoisomer at stimulating inositol phospholipid hydrolysis. 6 These results suggest that in the hamster urinary bladder, tachykinin‐induced inositol phospholipid breakdown is mediated via tachykinin receptors of the SP‐E type, as opposed to the SP‐P type.

N‐Methyl‐d‐Aspartate Receptor Desensitisation Is Neuroprotective by Inhibiting Glutamate‐Induced Apoptotic‐Like Death

Abstract: Glutamate excitotoxicity is implicated in several neurodegenerative diseases; consequently, considerable effort has been made to elucidate neuroprotective mechanisms against such toxicity. N‐Methyl‐d‐aspartate (NMDA) receptor desensitisation is one potential mechanism for controlling glutamate‐mediated neuronal cell death. Pretreatment of rat cerebellar granule cells with subtoxic concentrations of NMDA caused a marked reduction in the calcium signals generated by subsequent glutamate stimulation, and, furthermore, this receptor desensitisation was coupled to a reduction in glutamate‐induced apoptotic‐like death. These effects were reduced by either d‐2‐amino‐5‐phosphonopentanoic acid, an NMDA receptor antagonist, or cyclosporin A, an inhibitor of calcineurin. Thus, the results support a role for receptor desensitisation in protection from glutamate‐mediated apoptotic‐like neuronal cell death.

Molecular mechanisms of benzodiazepine‐induced down‐regulation of GABAA receptor α1 subunit protein in rat cerebellar granule cells

1 Chronic benzodiazepine treatment of rat cerebellar granule cells induced a transient down‐regulation of the γ‐aminobutyric acidA (GABAA) receptor α1 subunit protein, that was dose‐dependent (1 nM‐1 μm) and prevented by the benzodiazepine antagonist flumazenil (1 μm). After 2 days of treatment with 1 μm flunitrazepam the α1 subunit protein was reduced by 41% compared to untreated cells, which returned to, and remained at, control cell levels from 4–12 days of treatment. Chronic flunitrazepam treatment did not significantly alter the GABAA receptor α6 subunit protein over the 2–12 day period. 2 GABA treatment for 2 days down‐regulates the α1 subunit protein in a dose‐dependent (10 μm‐1 mM) manner that was prevented by the selective GABAA receptor antagonist bicuculline (10 μm). At 10 μm and 1 mM GABA the reduction in α1 subunit expression compared to controls was 31% and 66%, respectively. 3 The flunitrazepam‐induced decrease in α1 subunit protein is independent of GABA, which suggests that it involves a mechanism distinct from the GABA‐dependent action of benzodiazepines on GABAA receptor channel activity. 4 Simultaneous treatment with flunitrazepam and GABA did not produce an additive down‐regulation of α1 subunit protein, but produced an effect of the same magnitude as that of flunitrazepam alone. This down‐regulation induced by the combination of flunitrazepam and GABA was inhibited by flumazenil (78%), but unaffected by bicuculline. 5 The flunitrazepam‐induced down‐regulation of α1 subunit protein at 2 days was completely reversed by the protein kinase inhibitor staurosporine (0.3 μm). 6 This study has shown that both flunitrazepam and GABA treatment, via their respective binding sites, caused a reduction in the expression of the GABAA receptor α1 subunit protein; an effect mediated through the same neurochemical mechanism. The results also imply that the benzodiazepine effect is independent of GABA, and that the benzodiazepine and GABA sites may not be equally coupled to the down‐regulation process, with the benzodiazepine site being the more dominant. The biochemical mechanism underlying the benzodiazepine‐mediated down‐regulation of the α subunit protein seems to involve the activity of staurosporine‐sensitive protein kinases.

Solubilisation of the γ-Aminobutyric Acid/Benzodiazepine Receptor from Rat Cerebellum: Optimal Preservation of the Modulatory Responses by Natural Brain Lipids

Abstract: We have solubilised the γ‐aminobutyric acid/ benzodiazepine (GABA/BDZ) receptor from rat cerebellum using 3‐[(3‐cholamidopropyl)dimethylammonio] l‐propane sulphonate (CHAPS) in the presence of a natural brain lipid extract and cholesteryl hemisuccinate. The soluble material shows a homogeneous [3H]flunitrazepam ([3H]FNZ) binding population with an equilibrium dissociation constant (KD) of 4.4 × 0.2 nM compared to a KD of 2.3 × 0.2 nM in cerebellar synaptosomal membranes. The receptor complex in solution retains the characteristic facilitation of [3H]flunitrazepam binding induced by GABA, the pyrazolopyridine cartazolate, and the depressant barbiturate pentobarbital to the same extent as that observed in synaptosomal membranes. Furthermore, these responses are retained both quantitatively and qualitatively when this preparation is stored for 48 h at 4°C. This is contrary to the results obtained with a CHAPS‐soluble preparation including asolectin in which these responses are anomalous and extremely labile on storage.

Ca2+ permeability and Joro spider toxin sensitivity of AMPA and kainate receptors on cerebellar granule cells

We have investigated the Ca2+ permeability of native kainate- and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate- (AMPA) receptors in cultured rat cerebellar granule cells. Intracellular Ca2+ ([Ca2+]i) increases and Mn2+ quench of fura-2 (a measure of Ca2+ entry) mediated by kainate receptors were completely dependent on the presence of extracellular Na+. Kainate receptor-mediated [Ca2+]i rises were reduced 37% by the L-type voltage-gated Ca2+ channel blocker nifedipine (1 microM). AMPA receptor-mediated [Ca2+]i rises observed in Na+-free buffer were sensitive to Joro spider toxin (500 nM) blockade showing a 65% reduction, while kainate receptor-mediated [Ca2+]i responses were largely insensitive. These results suggest that a component of AMPA receptor-mediated [Ca2+]i increases occurs through Ca2+ permeable receptors which lack the GluR2 subunit and are Joro spider toxin sensitive. In contrast, kainate receptors do not appear to directly gate significant Ca2+ but raise [Ca2+]i through activation of voltage-gated Ca2+ channels and seem largely insensitive to Joro spider toxin.

The performance of junior doctors in applying clinical pharmacology knowledge and prescribing skills to standardized clinical cases

AIMS Recent studies suggest a worryingly high proportion of final year medical students and new doctors feel unprepared for effective and safe prescribing. Little research has been undertaken on UK junior doctors to see if these perceptions translate into unsafe prescribing practice. We aimed to measure the performance of foundation year 1 (FY1) doctors in applying clinical pharmacology and therapeutics (CPT) knowledge and prescribing skills using standardized clinical cases. METHODS A subject matter expert (SME) panel constructed a blueprint, and from these, twelve assessments focusing on areas posing high risk to patient safety and deemed as essential for FY1 doctors to know were chosen. Assessments comprised six extended matching questions (EMQs) and six written unobserved structured clinical examinations (WUSCEs) covering seven CPT domains. Two of each assessment types were administered over three time points to 128 FY1 doctors. RESULTS The twelve assessments were valid and statistically reliable. Across seven CPT areas tested 51-75% of FY1 doctors failed EMQs and 27-70% failed WUSCEs. The WUSCEs showed three performance trends; 30% of FY1 doctors consistently performing poorly, 50% performing around the passing score, and 20% performing consistently well. Categorical rating of the WUSCEs revealed 5% (8/161) of scripts contained errors deemed as potentially lethal. CONCLUSIONS This study showed that a large proportion of FY1 doctors failed to demonstrate the level of CPT knowledge and prescribing ability required at this stage of their careers. We identified areas of performance weakness that posed high risk to patient safety and suggested ways to improve the prescribing by FY1 doctors.

Predicting medical student performance from attributes at entry: a latent class analysis

Medical Education 2011: 45: 308–316

Desensitization of histamine H1 receptor-mediated inositol phosphate production in HeLa cells.

1 Histamine stimulated the accumulation of total [3H]‐inositol phosphates (IPn) in control HeLa cells with an EC50 of 3.7 ± 0.7 μm in the presence of 10 mm LiCl. The maximum response to histamine after 15 min incubation was 43 ± 5% over basal accumulation and occurred at a concentration of 1 mm histamine. 2 The histamine‐induced IPn production in HeLa cells was confirmed as H1 receptor‐mediated, since the H1 antagonist mepyramine (10−6 m) inhibited the histamine response (10−4 m) by 83 ± 7%, whereas the H2 antagonist, ranitidine (10−4 m), and H3 antagonist, thioperamide (10−6 m), were ineffective. 3 Histamine (10−4 m) pretreatment of HeLa cells for 30 min desensitized the subsequent histamine‐induced IPn accumulation. The desensitized cells accumulated IPn in response to histamine with an EC50 of 1.7 ± 0.7 μm after 15 min incubation. The maximum histamine‐induced IPn accumulation at 10−4 m was 19 ± 5% over basal and was significantly lower (P < 0.03) than the maximum response in control cells. 4 The desensitization of histamine‐induced IPn accumulation was time‐dependent and, at a desensitizing histamine concentration of 10−4 m, the half‐maximal attenuation occurred after approximately 9 min and maximum desensitization was achieved by 15–20 min. The desensitization of the IPn accumulation was a reversible phenomenon and full recovery of the response occurred 150 min after the removal of the desensitizing histamine‐containing medium. The half‐time for the recovery of the histamine‐induced response was estimated at 120 min. 5 Bradykinin stimulated IPn accumulation in HeLa cells, and the EC50 in control cells of 1.9 ± 0.2 nm was not significantly different from the EC50 value from histamine‐pretreated cells of 1.6 ± 0.9 nm. The bradykinin response at 1 μm was 194 ± 48% over basal IPn accumulation in control cells and this value was significantly different (P < 0.04) from the 1 μm bradykinin‐induced IPn accumulation in histamine‐pretreated HeLa cells of 143 ± 38% over basal. 6 NaF stimulated IPn accumulation in control HeLa cells in a dose‐related manner, with the maximum effect occurring at 15–20 mm. The EC50 value for NaF‐stimulated IPn accumulation in control cells was 10.5 ± 1.1 mm and the maximum response was 136 ± 41% over basal after 20 min incubation. In histamine desensitized HeLa cells the EC50 value for NaF was 12.3 ± 0.4 mm after 20 min stimulation, which was not significantly different from the value obtained in control cells. The maximum NaF‐stimulated IPn formation in desensitized cells of 68 ± 23% over basal occurred at 15–20 mm and was significantly lower (P < 0.01) than that obtained in control cells. 7 We show here that the acute histamine pretreatment of HeLa cells results in the desensitization of histamine H1 receptor‐mediated IPn production. The desensitization was not restricted to the H1 receptor‐mediated signal transduction pathway, but also includes both the bradykinin‐ and NaF‐mediated responses, supporting a heterologous desensitization mechanism. Our results are consistent with the site of attenuation being at or distal to the G‐protein and the underlying mechanism being a slowed time‐course for the production of inositol phosphates.

Biochemical Characterization of an Isolated and Functionally Reconstituted γ-Aminobutyric Acid/Benzodiazepine Receptor

Abstract: We have solubilized, affinity‐purified, and functionally reconstituted the γ‐aminobutyric acid/benzodiazepine (GABA/BDZ) receptor from rat brain into natural brain lipid liposomes. The detergent, 3‐[(3‐cholamidopropyl)‐dimethylammonio] 1‐propanesulphonate, was employed for the isolation of the receptor in the presence of a whole rat brain lipid extract supplemented with cholesteryl hemisuccinate. The soluble and reconstituted protein showed a homogeneous [3H]flunitrazepam binding population and the allosteric modulation of this binding site by GABA, by the pyrazolopyridine, cartazolate, and by the depressant barbiturate, pentobarbital. The purified GABA/BDZ receptor when incorporated into liposomes has been visualized by electron microscopy and reveals rosette structures, 8–9 nm in diameter, which appear to have a central pore. Sodium dodecyl sulphate‐polyacrylamide gel electrophoresis of the reconstituted GABA/BDZ receptor reveals three major protein bands of 41, 52–56, and 59–62 kDa, the latter two of which appear as doublets. Functional receptor reconstitution is demonstrated by the measurement of GABA‐stimulated 36Cl‐ flux into the purified GABA/BDZ receptor incorporated liposomes and its modulation by the BDZs, barbiturates, and pyrazolopyridines.