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Biomedical subjects

I L Martin

Publications and source records attributed to I L Martin.

At least 55 records · Page 3Linked to original sources
PubMed1987

Polyclonal antibodies to agonist benzodiazepines.

Benzodiazepine-binding, immunoglobulin G class antibodies have been raised in three rabbits immunised with a conjugate of kenazepine coupled to keyhole limpet haemocyanin. The antibodies were assayed by [3H]flunitrazepam binding, followed by adsorption onto Staphylococcus aureus cells. Measurement of the rates of association and dissociation of [3H]flunitrazepam binding, together with saturation analysis of equilibrium binding, revealed varying degrees of heterogeneity in the affinity constants of the three rabbit antisera (equilibrium KD values 0.18 to 4.13 nM at 20-22 degrees). Specificity of the antibodies was investigated by testing a wide variety of compounds (at concentrations of up to 10-100 microM) for their ability to inhibit [3H]flunitrazepam binding. Only benzodiazepines known to act as agonists at their receptor sites in the central nervous system (CNS) caused an inhibition of binding. The rank orders of the IC50 values of these drugs for inhibition of [3H]flunitrazepam binding to IgG from two out of the three rabbits correlated significantly with that previously published for displacement of CNS receptor binding. The agonist beta-carboline derivative ZK 93423, the anxiolytic cyclopyrrolones suriclone and zopiclone and the purines inosine and hypoxanthine all failed to inhibit antibody binding, supporting previous suggestions that these drugs may bind at non-benzodiazepine recognition sites on the CNS receptor. The antibodies described are expected to provide useful reagents for raising anti-idiotypic antibodies directed against the CNS receptor and for the identification and purification of possible endogenous benzodiazepine receptor agonists in the CNS.

Animals↗
PubMed1987

Modulation of [3H]flunitrazepam binding to rat cerebellar benzodiazepine receptors by phosphatidylserine.

The influence of phosphatidylserine on ligand binding to the benzodiazepine/GABA receptor complex was assessed in rat cerebellar synaptic membranes and in a detergent-solubilized membrane preparation. Intact synaptic membranes or membranes solubilized with the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]propanesulfonate) were incubated with a range of concentrations of phosphatidylserine for 2 h at 4 degrees C, prior to use in radioligand binding assays. Phosphatidylserine, an endogenous membrane phospholipid, facilitated the site-specific binding of [3H]flunitrazepam to synaptic membranes and CHAPS-solubilized preparations. In addition, phosphatidylserine inhibited the facilitation of [3H]flunitrazepam binding induced by either cartazolate or gamma-aminobutyric acid (GABA). Although the maximum effect (38% facilitation of [3H]flunitrazepam binding; greater than 90% inhibition of the cartazolate action) was produced using 130 microM phosphatidylserine, a significant enhancement of [3H]flunitrazepam binding could be observed upon preincubation of synaptic membranes with concentrations of phosphatidylserine as low as 5 microM. These results suggest that endogenous phosphatidylserine may play a role in the regulation of benzodiazepine/GABA receptor function, possibly through modulation of the mechanisms which functionally link the various components of this complex receptor system.

Animals↗
PubMed1987

Evidence of strain differences in GABA-benzodiazepine coupling.

The effects of a single dose of oxazepam on seizure threshold, receptor occupancy and brain oxazepam concentration were investigated at several time points after drug treatment in two inbred strains of mice (NIH and C3H/HE). The C3H/HE strain showed a greater sensitivity to the effects of both pentylenetetrazol and oxazepam. Furthermore, the C3H/HE strain showed decreases in both receptor occupancy and seizure threshold across time, whereas the NIH strain showed no change in either measure. Although within-strain correlations were observed between seizure threshold and receptor occupancy, the C3H/HE strain had similar seizure thresholds to the NIH strain throughout but lower percentage receptor occupancies, thus a between-strain correlation was not observed. The C3H/HE strain had a higher number of specific benzodiazepine binding sits and these results may reflect a strain difference in GABA-benzodiazepine receptor coupling. In a further experiment, the development of acute tolerance to the effects of oxazepam was investigated. Brain concentrations of oxazepam and receptor occupancies were determined for each strain of mouse, at two different time points (1.5 and 7.5 h after drug treatment), at which equivalent seizure thresholds were obtained by manipulating the starting dose of oxazepam. For each strain, when equivalent seizure thresholds were observed at different time points, equivalent receptor occupancies were also observed. However, a trend towards higher brain concentrations of oxazepam at the later time point was detected for the two strains, suggesting that there may be some decrease across time in the affinity of the receptor for oxazepam.

Animals↗
PubMed1987

The benzodiazepines and their receptors: 25 years of progress.

The benzodiazepines were introduced into clinical practice in 1960 and since that time have become one of the most frequently prescribed of all psychotropic agents. Over the past 25 years the mechanism by which these compounds produce their effects has been revealed by a combination of electrophysiological and biochemical studies. It is now apparent that these drugs interact with a specific recognition site on the GABAA receptor complex and thereby modulate the gating of the chloride channel associated with this major inhibitory amino acid transmitter receptor. This modulation is novel in that while the clinically prescribed benzodiazepines increase the chloride current in response to a given GABA stimulus, agents are now known which produce the opposite effect. However, the details of the molecular mechanisms by which these events occur remain to be elucidated. Our progress toward this goal will rely on the acquisition of structural information about this receptor protein using the techniques of molecular biology and its marriage with new initiatives concerning the detail of ligand recognition. The interpretation of such information in the context of the complex pharmacology of this important receptor system is indeed a stimulating prospect.

Animals↗
PubMed1987

Solubilisation of the gamma-aminobutyric acid/benzodiazepine receptor from rat cerebellum: optimal preservation of the modulatory responses by natural brain lipids.

We have solubilised the gamma-aminobutyric acid/benzodiazepine (GABA/BDZ) receptor from rat cerebellum using 3-[(3-cholamidopropyl)dimethylammonio] 1-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 degrees 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.

Animals↗
PubMed1987

Solubilisation of the benzodiazepine receptor from rat cerebellum by the detergent n-octylpentaoxyethylene.

The detergent n-octylpentaoxyethylene is one in the series of tenside detergents developed for membrane solubilisation. We have used this detergent to solubilise benzodiazepine receptors from rat cerebellum. The soluble receptor has an affinity (KD) for [3H]flunitrazepam of 1.8 nM +/- 0.2, which is not significantly different from that observed in synaptic membranes. Under optimal conditions (0.6% wt/vol), the number of soluble flunitrazepam binding sites (Bmax) of 0.35 pmol/mg protein suggests an apparent solubilisation of 40% of sites from the membrane. However, the absence of the characteristic facilitation of [3H]flunitrazepam binding by gamma-aminobutyric acid (GABA), cartazolate, and pentobarbital in this soluble receptor preparation suggests that such a preparation is unlikely to be a useful preparation for further studies on the molecular mechanisms underlying GABAA receptor function.

Animals↗
PubMed1986

Solubilization of the benzodiazepine/gamma-aminobutyric acid receptor complex: comparison of the detergents octylglucopyranoside and 3-[(3-cholamidopropyl)-dimethylammonio]1-propanesulfonate (CHAPS).

Octylglucopyranoside (OCTG) was three times more efficient than 3-[(3-cholamidopropyl)-dimethylammonio]1-propanesulfonate (CHAPS) in solubilizing the benzodiazepine (BDZ)/gamma-aminobutyric acid (GABA) receptor complex from rat cerebellar synaptic membranes. OCTG-solubilized receptor preparations had ligand binding characteristics that were significantly different from those of the CHAPS-solubilized receptors. The inclusion of phospholipids in the solubilization media improved the binding characteristics of both soluble receptor preparations and appeared absolutely necessary for the maintenance of chloride facilitation of flunitrazepam (FNZ) binding to OCTG-solubilized receptors. FNZ and ethyl-beta-carboline-3-carboxylate bound to OCTG-solubilized preparations with equilibrium dissociation constants of 2.2 nM and 1.6 nM, respectively, and chloride (150 mM) and GABA (100 microM) + chloride facilitated the binding of FNZ by 15% and 55%, respectively; these ligand binding characteristics are similar to those of membrane-located BDZ receptors. Cartazolate, a pyrazolopyridine that facilitated the binding of FNZ to membrane-located and CHAPS-solubilized receptors, did not facilitate FNZ binding to OCTG-solubilized receptors. These results are discussed in terms of an interaction between the membrane lipid phosphatidylserine (PS) and cartazolate; PS appears to have the capacity to inhibit the effects of cartazolate on FNZ binding. Storage of the soluble receptor preparations for 24 h at 4 degrees C resulted in the loss of several characteristic BDZ receptor binding properties. Incorporation of the OCTG-solubilized receptor complex into liposomes prevented these losses but this procedure did not protect the CHAPS-solubilized receptors. We conclude that OCTG may have some advantages over CHAPS as the detergent of choice for the solubilization and reconstitution in liposomes of a functional BDZ/GABA receptor-chloride ionophore complex.

Animals↗
PubMed1985

Long-lasting anticonvulsant effects of diazepam in different mouse strains: correlations with brain concentrations and receptor occupancy.

There were marked strain differences in the duration of the protective effects of diazepam against the convulsant actions of penylenetetrazole and picrotoxin in mice. In no case was significant protection found at 12 h or longer, regardless of whether the incidence of or the latencies to myoclonus or tonic-clonic convulsions were considered. These behavioural differences could not be explained simply in terms of strain differences in benzodiazepine metabolism or in percent of receptor occupancy, as determined by the fractional displacement of 3H-flunitrazepam binding in vivo. It is suggested that there might be strain differences in the percent of receptor occupancy needed in order to produce an anticonvulsant effect.

Animals↗
PubMed1984

Modification of pyrazoloquinolinone affinity by GABA predicts efficacy at the benzodiazepine receptor.

A series of pyrazoloquinolinones CGS 9896, CGS 9895 and CGS 8216 have been reported to exhibit agonist, partial agonist and antagonist properties, respectively, at the benzodiazepine receptor. We have examined the effects of these compounds and of diazepam on pentylenetetrazole seizure thresholds in mice and found CGS 9896 to be a partial agonist and CGS 8216 a weak inverse agonist in this respect; CGS 9895 was essentially devoid of efficacy. In the presence of 100 microM GABA and 150 mM NaCl, the affinity of CGS 9896 for the benzodiazepine receptor at 37 degrees C was significantly increased whereas the affinity of CGS 8216 was similarly decreased and that of CGS 9895 was unchanged. The effect of GABA on the affinity of these ligands for the benzodiazepine receptor at 37 degrees C was thus predictive of the pharmacological efficacy which we observed.

Animals↗
PubMed1984

Photoaffinity labelling of the benzodiazepine receptor compromises the recognition site but not its effector mechanism.

When subjected to ultraviolet light flunitrazepam could be irreversibly attached to brain membrane preparations such that the affinity of the benzodiazepine receptor for certain ligands, such as diazepam, becomes markedly reduced. However, the apparent affinity for diazepam is increased by the addition of gamma-aminobutyric acid and sodium chloride, to the same extent in this membrane preparation as in membranes not so pretreated. This observation suggests that photoaffinity labelling of the benzodiazepine receptor with flunitrazepam modifies the recognition characteristics of the receptor but not its effector mechanism.

Affinity Labels↗
PubMed1984

Kinetics of [3H]Ro 15-1788 binding to membrane-bound rat brain benzodiazepine receptors.

Ro 15-1788 is thought to interact specifically with the benzodiazepine receptor population in the mammalian CNS as an antagonist. We have compared the kinetics of interaction of this ligand with the benzodiazepine agonist flunitrazepam in the rat cerebellum. The association of [3H]Ro 15-1788 with the benzodiazepine receptor in this brain region is monoexponential, and the dissociation, initiated either by dilution or by displacement with a number of different ligands, also obeys simple monoexponential kinetics. There is no evidence of cooperative interactions with this ligand, and its dissociation is unaffected by the presence of 100 microM gamma-aminobutyric acid and/or 150 mM sodium chloride. In contrast, the dissociation of the agonist [3H]flunitrazepam is biphasic, and the possible interpretation of this data in terms of agonist-induced conformational change is discussed. Evidence is also presented that the mechanism of interaction of Ro 15-1788 with the benzodiazepine receptor population in hippocampal membranes is distinct from that found in the cerebellum.

Animals↗
PubMed1983

Photoaffinity labelling of the benzodiazepine receptor cannot be used to predict ligand efficacy.

Irreversible attachment of flunitrazepam to brain membranes occurs on exposure to UV light, resulting in a loss of the number of binding sites detected by saturation binding of [3H]flunitrazepam, but not of [3H]ethyl-beta-carboline-3-carboxylate (beta CCE). Changes in the affinity of ligands for the benzodiazepine receptor produced by photoaffinity labelling, as indicated by the relative IC50 values for the displacement of [3H]beta CCE in photoaffinity labelled versus control membranes, appear to be related to their chemical structure rather than to their pharmacological profile.

Animals↗
PubMed1983

The benzodiazepine receptor in rat brain and its interaction with ethyl beta-carboline-3-carboxylate.

[3H]Ethyl beta-carboline-3-carboxylate ( [3H] beta-CCE) binds to a homogeneous population of recognition sites in rat whole brain membranes with high affinity. The [3H]beta-CCE binding is completely displaceable by low concentrations of a number of benzodiazepines with similar potencies found when using a 3H-benzodiazepine as the ligand. This suggests that the recognition sites for beta-CCE and the benzodiazepines are identical or that they are involved in a close interaction. The binding of [3H]beta-CCE does not obey simple mass-action kinetics. [3H]Flunitrazepam dissociation from its receptor population is biphasic, and different methods of initiation of this dissociation indicate that cooperative interactions take place within the receptor population. We conclude that the benzodiazepine receptor is a single entity that can exist in two conformations, the equilibrium between which may be controlled by some as yet unidentified factor.

Animals↗