Manfred Karobath

Calcium antagonist binding sites in the rat brain: Quantitative autoradiographic mapping using the 1,4-dihydropyridines [3H]PN 200-110 and [3H]PY 108-068

Anin vitro autoradiographic technique has been used for the quantitative mapping of calcium antagonist binding sites (CABS) in the rat brain, using the 1,4-dihydropyridines [3H]PN 200-110 and [3H]PY 108-068 as ligands. CABS were distributed throughout the brain in a highly heterogeneous fashion. The highest densities of CABS were observed in the olfactory bulb, hippocampus and parts of the amygdala. The neocortex was also rich in CABS. The basal ganglia, thalamus and hypothalamus presented intermediate levels of CABS while low densities of sites were seen in areas such as the cerebellum, pons and white matter tracts. The distributions of CABS in brain does not correlate with indexes of brain blood flow, regional glucose utilization or the distributions of receptor binding sites for drugs and neuro-transmitters analyzed until now. No correlation exists between CABS distribution and that of any neurotransmitter or brain enzyme described so far. The heterogeneous distributions of CABS is suggestive of a neuronal localization, an idea supported by lesion experiments.

The effects of lesions in the rat hippocampus suggest the association of calcium channel blocker binding sites with specific neuronal population.

The binding of calcium antagonists in the rat hippocampal formation was studied using autoradiography. Hippocampal slices were labeled in vitro with [3H]PN 200-110. High densities of binding sites for calcium antagonists were found in the molecular layer of the dentate gyrus and in the CA3 subfield of the hippocampus. After ablation of the granule cells by local injection of colchicine a marked decrease in the number of [3H]PN 200-110 binding sites density was observed on these areas, while binding to other parts of the hippocampal formation and brain was spared. These results strongly suggest the localization of high densities of calcium channels to the granule cells of the dentate gyrus.

Taurine uptake in synaptosomal fractions of rat cerebral cortex.

Abstract— [35S]Taurine was found to be accumulated in synaptosomal fractions of rat cerebral cortex. Kinetic analysis in the range of 1–800 μm‐[35S]taurine revealed at least two different uptake processes. A high affinity uptake with a Km of 20 μM and a low affinity uptake with a Km of about 450 μM. The high affinity component was dependent on temperature and energy, and virtually abolished in the absence of sodium. Examination of the influence of structural analogues and putative transmitter substances indicates that the high affinity uptake of taurine into synaptosomal fractions of rat cerebral cortex is unique and highly specific. No specific actions of several centrally acting drugs on taurine uptake could be observed.

In vitro modulations by SQ 20009 and SQ 65396 of GABA receptor binding in rat CNS membranes

SQ 20009 and SQ 65396, two new pyrazolopyridines with anxiolytic properties in animals, reversibly increased Na+-independent 3H-GABA binding to rat cerabral cortex membranes in vitro. This modulation was partially chloride dependent and sensitive to Triton-X 100. Kinetic analysis of 3H-muscimol binding revealed an increase of the apparent number of binding sites. The results are compatible with a functional association between Na+-independent GABA binding sites and the GABA/benzodiazepine receptor complex.

The interaction of [3H]PY 108-068 and of [3H]PN 200-110 with calcium channel binding sites in rat brain

The binding properties of the tritiated calcium channel antagonists PY 108-068 and PN 200-110 were investigated in membrane fractions from rat brainin vitro. Both ligands reversibly interact with one apparent population of stereoselective binding sites which have pharmacological properties described for calcium channel binding sites. In a calcium buffer enhancement of [3H]PY 108-068 binding is observed with an EC50 at pCa 6.28 [3H]PN 200-110 binding is less sensitive to allosteric stimulation by diltiazem and to allosteric inhibition by verapamil and D 600 than [3H]PY 108-068 binding, suggesting that the former ligand may stabilize a high affinity configuration of the binding sites. Afteri.v. administration of [3H]PY 108-068in vivo binding to membranes is observed in brain and heart, which, in contrast to total tissue radioactivity is sensitive to inhibition by unlabelled (+) PN 200-110. These observations suggest that PY 108-068 can interact with its binding sites alsoin vivo. The results ofex vivo binding studies in brain and heart with [3H]PY 108-068 confirm and extend these observations. It could be shown that all investigated 1,4-dihydropyridines (PY 108-068, PN 200-110, nifedipine, Bay K 8644) after i.p. administration can readily enter brain and heart tissue.

Distinction of benzodiazepine agonists from antagonists by photoaffinity labelling of benzodiazepine receptors in vitro.

When membranes of rat cerebellum are exposed to UV light in the presence of flunitrazepam this ligand can be incorporated into one of the assumed 4 benzodiazepine binding sites of the GABA-benzodiazepine receptor complex. This irreversible incorporation of flunitrazepam, in contrast to reversible binding of this substance, leads to conformational changes of the remaining 3 benzodiazepine binding sites which result in a decreased affinity of benzodiazepine agonists, but not of benzodiazepine antagonists. The investigation of the affinity of drugs for [3H]benzodiazepine antagonist binding before and after photoaffinity labelling of benzodiazepine receptors with flunitrazepam can therefore be used as a sensitive and simple test to distinguish between agonists and antagonists in vitro.

Modulation of GABA binding sites by CNS depressants and CNS convulsants.

[(3)H]Muscimol binding at 23 degrees C and muscimol stimulated [(3)H]flunitrazepam binding at 37 degrees C to membranes of rat cerebral cortex have been investigated. In washed membrane preparations, 2 apparent populations of [(3)H]muscimol binding sites can be observed. At 23 degrees C [(3)H]muscimol binding is more sensitive to inhibition by NaCl and by other salts than at 0 degrees C. The CNS depressants etazolate and pentobarbital reversibly enhance [(3)H]muscimol binding and they increase the affinity of muscimol as a stimulator of [(3)H]flunitrazepam binding. Conversely the CNS convulsants picrotoxin, picrotoxinin and isopropylbicyclophosphate (IPTBO) reversibly interfere with [(3)H]muscimol binding when NaCl is present and these drugs antagonize the effects of etazolate. In the presence of NaCl, picrotoxin, picrotoxinin and IPTBO also decrease the apparent affinity of muscimol or GABA as stimulator of [(3)H]flunitrazepam binding. Binding of [(3)H]muscimol to GABA recognition sites of rat cerebral cortex is enhanced by Ag(+), Hg(+) and Cu(2+) in ?M concentrations, Ag(+) being most potent. The effects of 100 ?M AgNO(3) persist after repeated washing of the membranes. When membranes are pretreated with AgNO(3) only one apparent population of [(3)H]muscimol binding sites with high affinity (K(d): 6-8 nM) is found. In AgNO(3) pretreated membranes, the affinity of muscimol as stimulator of [(3)H]flunitrazepam binding is increased 18 times (EC(50) 14 nM) when compared to control membranes, (EC(50) 253 nM). In AgNO(3) pretreated membranes, etazolate, pentobarbital and IPTBO fail to perturb either [(3)H]muscimol binding or baseline and muscimol stimulated [(3)H]flunitrazepam binding. The results demonstrate that the apparent sensitivity of GABA binding sites of the GABA-benzodiazepine-picrotoxin receptor complex can be increased by etazolate and pentobarbital and decreased by picrotoxin and IPTBO. These drugs have in common that they interfere with [(3)H]dihydropicrotoxinin binding.

Modulation by picrotoxin and IPTBO of 3H-flunitrazepam binding to the GABA/benzodiazepine receptor complex of rat cerebellum

Abstract Picrotoxin and IPTBO, two CNS convulsants which block chloride permeability of GABA receptor regulated chloride ion channels, have modulatory effects on 3 H-flunitrazepam binding to the GABA/benzodiazepine receptor complex of the cerebellum. Their actions on 3 H-flunitrazepam binding to membranes from rat cerebellum include: (i) both drugs in high concentrations stimulate 3 H-flunitrazepam binding mainly by increasing the apparent number of binding sites. This effect is chloride ion dependent and reversible; (ii) they inhibit 3 H-flunitrazepam binding stimulated by the pyrazolopyridine etazolate (SQ 20009), and this effect is observed in binding experiments performed at low (0 °C) and at physiological (35 °C) temperature; (iii) they inhibit GABA stimulated binding in a chloride ion dependent mode. This action is much more potent when the binding experiments are performed at physiological temperatures. These results demonstrate that drugs which act on GABA receptor associated chloride ion channels have potent modulatory effects on benzodiazepine receptor binding in cerebellum.

Evidence for specific synaptosomal localization of exogenous accumulated taurine.

Abstract— Equilibrium or incomplete equilibrium density gradient centrifugation was used to characterize the subcellular localization of exogenous [3 5S]taurine which was taken up by minces or homogenates of rat cerebral cortex. [3 5S]Taurine is accumulated in synaptosomes, which sediment more slowly than l‐[3H]norepinephrine‐accumulating particles. When [3 5S]taurine and [3H]GABA are accumulated by minces, a small difference in the sedimentation profile of taurine and GABA was observed, but no difference was found when taurine and intrasynaptosomal potassium were compared. However, potassium sedimented more slowly after incubation of homogenates than of minces. These data give evidence for the accumulation of [3 5S]taurine by a specific synaptosomal population.

The effect of temperature and chloride ions on the stimulation of [3H]Flunitrazepam binding by the muscimol analogues THIP and piperidine-4-sulfonic acid

THIP and piperidine-4-sulfonic acid (PSA) interact with [3H]GABA binding sites and have GABAmimetic efficacy in vivo, but fail to enhance benzodiazepine receptor binding performed at 0 degree C. However, when [3H]flunitrazepam binding is determined at elevated temperature (30 or 37 degrees C), THIP and PSA display potent chloride ion-dependent stimulatory effects. These results resolve apparent discrepancies between the properties of GABA receptors observed in vivo and in vitro, and they suggest that the modulation of benzodiazepine receptor binding investigated at physiological temperatures can be used as an experimental system for the characterization of GABA receptors.