A. Uzan

Labelling of “Peripheral‐Type” Benzodiazepine Binding Sites in the Rat Brain By Using [3H]PK 11195, an Isoquinoline Carboxamide Derivative: Kinetic Studies and Autoradiographic Localization

Abstract: PK 11195 [1‐(2‐chlorophenyl)‐N‐methyl‐N‐(1‐methylpropyl)‐3‐isoquinolinecarboxamide] is a new ligand for the “peripheral‐type” benzodiazepine binding sites, chemically unrelated to benzodiazepines. It displaces with a very high potency (IC50× 10−9M) [3H]‐RO5–4864 (a benzodiazepine which specifically labels the peripheral‐type sites) from its binding sites. [3H]PK 11195 binds to a membrane fraction from rat brain cortex and rat olfactory bulb in a saturable and reversible manner with a very high affinity (KD= 10−9M). The number of maximal binding sites was ten times greater in the olfactory bulb than in the brain cortex. The order of potency of several compounds as displacers at 25°C (PK 11195 > RO5–4864 > diazepam > dipyridamole > clonazepam) demonstrates that [3H]PK 11195 binds to the peripheral‐type benzodiazepine binding sites. The KD value for the [3H]PK 11195 binding is not affected by temperature changes, whereas RO5–4864 and diazepam affinities decrease with increasing temperatures. Autoradiographic images of [3H]PK 11195 binding to rat brain sections show that binding sites are mainly localized in the olfactory bulb, median eminence, choroid plexus, and ependyma. This ligand could be a useful tool to elucidate the physiological and pharmacological relevance of these binding sites.

Differentiation between two ligands for peripheral benzodiazepine binding sites, [3H]R05-4864 and [3H]PK 11195, by thermodynamic studies

The [3H]PK 11195, 1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinolinecarboxamide, binding sites in rat cardiac membranes are saturable, with high affinity, specific GABA-independent and correspond to the peripheral type of benzodiazepine. The order of potency of displacing agents was: PK 11195 greater than RO5-4864 greater than dipyridamole greater than diazepam greater than clonazepam. The Bmax obtained with [3H]PK 11195 was equivalent of the Bmax obtained with [3H]RO5-4864 in the same experimental conditions. However thermodynamic analysis indicates that the [3H]PK 11195 binding was entropy driven whereas the [3H]RO5-4864 binding was enthalpy driven. Consequently PK 11195 might be an antagonist of these binding sites and RO5-4864 an agonist or a partial agonist. The simultaneous use of both drugs might help to elucidate the physiological relevance of peripheral benzodiazepine binding sites.

2-Amino-6-trifluoromethoxy benzothiazole, a possible antagonist of excitatory amino acid neurotransmission—I

2-Amino-6-trifluoromethoxy benzothiazole (PK 26124) prevented convulsions induced in rodents by maximal electroshock, inhibitors of the synthesis of gamma-aminobutyric acid (GABA) and ouabain, but was inactive against seizures provoked by GABA antagonists, unlike diazepam, chlordiazepoxide, phenobarbital and valproic acid. 2-Amino-6-trifluoromethoxy benzothiazole prevented seizures induced by sound stimuli in DBA/2 mice (ED50 = 0.66; 2.1 and 4.1 mg/kg, i.p. according to the seizure component), postural seizures in El mice (ED50 = 7.5 mg, i.p.) and seizures induced by photic stimulation in the baboon, Papio papio, at 4 and 8 mg/kg (i.v.). This spectrum of anticonvulsant activity closely resembles that reported previously for dicarboxylic amino acid antagonists. Indeed, PK 26124 prevented seizures induced by L-glutamate (ED50 = 8.5 mg/kg, i.p.) or by kainate (ED50 = 9.25 mg/kg, i.p.) and tremors induced by harmaline (ED50 = 2.5 mg/kg, i.p.) In these tests diazepam was inactive (L-glutamate) or as potent as PK 26124 (kainate, harmaline), whereas it was 10-20 times more potent than PK 26124 against seizures induced by inhibitors of the synthesis of GABA. Together, these data suggest that PK 26124 possesses antagonistic properties of excitatory dicarboxylic amino acids, which may contribute to its anticonvulsant action.

2-amino-6-trifluoromethoxy benzothiazole, a possible antagonist of excitatory amino acid neurotransmission. II: biochemical properties

Two models have been chosen to study the effect of 2-amino-6-trifluoromethoxy benzothiazole (PK 26124) on excitatory amino acid neurotransmission: the pool of cyclic guanosine monophosphate (cGMP) in the cerebellum and the release of acetylcholine in the striatum and olfactory tubercles. The release of acetylcholine induced by N-methyl-DL-aspartate in the striatum and olfactory tubercles was antagonized by PK 26124 which was less potent on the release of acetylcholine induced electrically. The increase in levels of cGMP in the cerebellum induced by excitatory amino acids such as glutamate and quisqualate was antagonized by PK 26124, but the drug was inactive against N-methyl-DL-aspartate, L-aspartate, kainate and cysteine sulphinate. In vivo it antagonized the increases of cGMP in the cerebellum elicited by all these excitatory compounds. All these results are compatible with a possible antagonism by PK 26124 of the excitatory amino acid neurotransmission and may explain its anticonvulsant properties.

Electrophysiological and pharmacological evidence that peripheral type benzodiazepine receptors are coupled to calcium channels in the heart.

PK 11195, an antagonist of the peripheral type benzodiazepine receptor, does not affect either the duration of the action potential or the tension of the guinea pig papillary muscle. However, it antagonized the effects of the calcium channel blockers, nitrendipine, verapamil, diltiazem, and of BAY K8644, a calcium channel agonist in this heart preparation. On the other hand, PK 11195 does not change the increase in the action potential duration provoked by the potassium channel blocker tetraethylammonium. RO5-4864, an agonist of the peripheral type benzodiazepine receptor, decreased the tension of the guinea pig papillary muscle. The effect was reversed by increasing extracellular Ca2+ concentrations up to 4 mM. These results suggest that in the heart the peripheral type benzodiazepine receptors are coupled to calcium channels.

Electrophysiological and pharmacological characterization of peripheral benzodiazepine receptors in a guinea pig heart preparation.

RO5-4864 decreased in a dose-dependent manner, from 3 X 10(-9) M to 3 X 10(-6) M, the duration of intracellular action potential and the contractility in a guinea pig preparation. Diazepam was less effective and clonazepam inactive. The effects of RO5-4864 were GABA-independent and antagonized by PK 11195 but not by the selective antagonist of the brain type benzodiazepine receptors RO15-1788. These results show the pharmacological relevance of peripheral type benzodiazepine binding sites at the cardiac level.

Labelling of peripheral-type benzodiazepine binding sites in human brain with [3H]PK 11195: Anatomical and subcellular distribution

The peripheral-type benzodiazepine binding site, erstwhile characterized in the rodent and feline brain, has now been characterized in post-mortem human brain using [3H]PK 11195. The kinetics and pharmacological properties of the binding of this ligand are similar to peripheral-type benzodiazepine binding sites elsewhere. The potency of RO5-4864 for this site in human brain is close to that seen in ruminant and carnivore tissues but considerably lower than in rodent tissues. The regional distribution of these binding sites would suggest a neuronal rather than a glial localization. [3H]PK 11195 bound in a similar fashion to slide-mounted sections of human brain, thus allowing quantitative studies of the regional distribution of peripheral-type benzodiazepine binding sites to be made. The binding sites were distributed heterogeneously, but were restricted to the grey matter. Highest densities of binding sites were found in forebrain structures. The localization was not limited to any functional system, nor did it resemble any previously described transmitter system. The similarities between peripheral-type benzodiazepine binding sites in human and in feline brain in terms of their pharmacological characteristics and their regional and subcellular distribution suggest that the cat, rather than the rat, may be the better model for studying a possible role for this site in human cerebral function.

Multiple benzodiazepine receptors: Evidence of a dissociation between anticonflict and anticonvulsant properties by PK 8165 and PK 9084 (two quinoline derivatives)

Abstract PK 8165 and PK 9084, two quinoline derivatives, displace [ 3 H]-diazepam and its binding sites. These drugs appeared to be more potent in the presence of halides suggesting that they are acting on benzodiazepine receptors associated to a chloride ionophore. Like the benzodiazepines PK 8165 and PK 9084 increase punished responding in the rat conflict procedure but do not produce ataxia or sedation even at doses 5 to 20 times higher than those which are effective in the conflict test. Moreover they do not possess anticonvulsant properties. PK 8165 and PK 9084 do not change cGMP content of the cerebellar cortex and do not antagonize the increase in cGMP induced by the GABA antagonists isoniazid and picrotozin. Thus PK 8165 and PK 9084 “pure anticonflict drugs” might act on GABA-independent benzodiazepine receptors associated to a chloride ionophore.

Identification of stereospecific [3H]spiroperidol binding sites in mammalian lymphocytes

Abstract Direct binding to intact rat lymphocytes has been shown for the potent dopaminergic antagonist [ 3 H]spiroperidol. The specific binding is saturable with two components (K D1 = 1.9 nM, K D2 = 36.2 nM). Determination of the K D by kinetic studies measuring rate constants for association and dissociation provided K D values similar to those obtained in equilibrium experiments. The specific binding is proportional to cell concentration and temperature dependent with a maximum at 37°C. [ 3 H]spiroperidol binding is stereospecific since (+)butaclamol was more effective than (−)butaclamol. The relative potencies of different antidopaminergic agents in competing for [ 3 H]spiroperidol binding sites parallel their activity in the striatum. Dopaminergic receptors have also been demonstrated in other mammalian lymphocytes (rabbit, dog, human). Lymphocyte dopaminergic receptors could be implicated in lymphocytes mediated immune response.

Dihydropyridine and peripheral type benzodiazepine binding sites: Subcellular distribution and molecular size determination

Electrophysiological and pharmacological studies have shown that peripheral-type benzodiazepine receptors modulate voltage-sensitive calcium channels in the heart. We have compared these binding sites with binding sites for [3H]dihydropyridines, which are believed to label such channels. Although no direct or allosteric interaction could be demonstrated between the two sites, their subcellular distribution--sarcolemma and ryanodine-sensitive sarcoplasmic reticulum--was parallel. Size determination of the two sites suggests that the receptors for these two classes of compounds are separate molecules packaged in the same membrane compartment.