The 5-hydroxytryptamine-releasing properties of two epimer quinoline derivatives.

Two epimer quinoline derivatives, PK 5078 and PK 7059, have been shown to be potent at releasing 5-HT from blood platelets. Moreover PK 5078 was also a potent and selective inhibitor of the uptake of 5-HT, being about 20 times as active as clomipramine. Both drugs, like p-chloroamphetamine, released 5-HT but did not inhibit MAO-A. Whilst p-chloroamphetamine seemed to be active on the cytoplasmic pool of 5-HT and reserpine on the vesicular pool, PK 5078 and PK 7059 were effective first on the vesicular pool and then on the cytoplasmic pool. The quinoline derivatives were devoid of the typical side-effects of amphetamine-like drugs, i.e. hyperactivity, anorexia and group toxicity. For these reasons PK 5078 and PK 7059 can be considered to be a new type of selective 5.HT-releasing drug.

Characterization of peripheral type benzodiazepine binding sites in human and rat platelets by using [3H]PK 11195. Studies in hypertensive patients.

Peripheral type benzodiazepine binding sites have been studied in human and rat platelets and platelet membranes by using PK 11195 (1-(2-chlorophenyl)-N-methyl-N-(1-methyl propyl)-3-isoquinolinecarboxamide) as a ligand. [3H]PK 11195 binding to the intact cells and membranes is saturable, with a high affinity and presents the pharmacological specificity corresponding to the peripheral binding sites (PK 11195 greater than RO5-4864 greater than diazepam greater than clonazepam). [3H]PK 11195 affinity is not affected by cell lysis, but there is a loss of binding capacity, contrarily to RO5-4864 whose affinity is greatly diminished. For this reason [3H]RO5-4864 binding can only be demonstrated in intact cells. Furthermore opposite to RO5-4864, PK 11195 affinity is not decreased by increasing temperatures. No difference was found between binding parameters (KD and Bmax) for [3H]PK 11195 between normotensive and hypertensive subjects. The very high binding capacity of human and rat platelets (Bmax greater than pmole/10(8) cells) makes them a good biological model for studying the physiological significance of "peripheral type" benzodiazepine binding sites.

PK 11195, an antagonist of peripheral benzodiazepine receptors, reduces ventricular arrhythmias during myocardial ischemia and reperfusion in the dog.

PK 11195, an antagonist of peripheral type benzodiazepine receptors, in doses from 5 to 25 mg/kg i.d. protected in a dose-dependent manner dogs against both early and delayed ventricular arrhythmias induced by 20 min ischemia and against ventricular fibrillation following reperfusion. Thus, peripheral-type benzodiazepine receptors might represent a novel target in the treatment of angina and cardiac ischemia.

Stereoselective inhibition of the binding of [3H]PK 11195 to peripheral-type benzodiazepine binding sites by a quinolinepropanamide derivative.

The specific binding of [3H]PK 11195 to the peripheral-type benzodiazepine binding site is inhibited by the l-enantiomer of N,N-diethyl-alpha-methyl-2-phenyl-4-quinolinepropanamide ((-)Q1) but not by its d-enantiomer ((+)Q1). (-)Q1 inhibited [3H]PK 11195 binding to several rat tissues with an IC50 of less than 10 nM whereas (+)Q1 was at least 500 times less potent. This stereoselectivity was observed in all the tissues tested (brain, heart, kidney and adrenals). The same stereoselectivity was found for the displacement of the binding of [3H]PK 11195 in vivo, where (-)Q1 had an ID50 between 4-15 mg/kg and (+)Q1 was completely inactive at all doses tested (i.e. up to 40 mg/kg). Neither isomer had appreciable affinity for central-type benzodiazepine binding sites ([3H]diazepam) nor for voltage-sensitive calcium channels ([3H]PN 200210 and [3H]verapamil).

In vivo labelling in several rat tissues of 'peripheral type' benzodiazepine binding sites.

'Peripheral type' benzodiazepine binding sites in several rat tissues were labelled by intravenous injection of [3H]PK 11195 and [3H] RO5 -4864. Binding was saturable in all tissues studied and regional distribution paralleled the in vitro binding. A similar potency order of displacing compounds was found in vivo and in vitro PK 11195 greater than PK 11211 greater than RO5 -4864 greater than diazepam greater than dipyridamole greater than clonazepam. These results demonstrate the feasibility of using this technique to examine the effects of pharmacological manipulation on the binding sites in their native state. However some properties (broader maximum during time course, higher percentage of particulate binding in the brain and independence of temperature) make [3H]PK 11195 the most suitable ligand for this kind of studies.

Autoradiographic localization of peripheral benzodiazepine binding sites in the cat brain with [3H]PK 11195.

"Peripheral type" benzodiazepine binding sites were labelled in cat brain membranes by using [3H]PK 11195. This ligand binds to the "peripheral type" binding sites in a reversible, specific and saturable manner. Cat brain binding sites density (congruent to 6 pmol/mg prot.) was higher than in the rat. Pharmacological specificity was demonstrated by the potency order of displacing agents: PK 11195 greater than RO5-4864 greater than dipyridamole greater than diazepam greater than clonazepam. A similar characterization was performed in slide mounted brain sections where [3H]PK 11195 also labelled the "peripheral type" benzodiazepine binding sites. The high percentage of specific binding (80%) at 1 nM of [3H]PK 11195 made possible the autoradiographic studies on binding sites distribution. These sites were heterogeneously distributed in the grey matter and absent in white matter. Visual, auditory and other specific sensory relay stations were highly labelled. The blood pressure regulating nuclei, the vestibulo-cerebellar and the extrapyramidal motor system also presented a very high binding density. As previously described in the rat brain, choroid plexus was also strongly labelled by [3H]PK 11195 in the cat.

Complex pharmacology of natural cannabinoids: evidence for partial agonist activity of delta9-tetrahydrocannabinol and antagonist activity of cannabidiol on rat brain cannabinoid receptors.

Delta9-tetrahydrocannabinol (delta9-THC), cannabinol and cannabidiol are three important natural cannabinoids from the Marijuana plant (Cannabis sativa). Using [35S]GTP-gamma-S binding on rat cerebellar homogenate as an index of cannabinoid receptor activation we show that: delta9-THC does not induce the maximal effect obtained by classical cannabinoid receptor agonists such as CP55940. Moreover at high concentration delta9-THC exhibits antagonist properties. Cannabinol is a weak agonist on rat cerebellar cannabinoid receptors and cannabidiol behaves as an antagonist acting in the micromolar range.

Opposite effects of two ligands for peripheral type benzodiazepine binding sites, PK 11195 and RO5-4864, in a conflict situation in the rat.

The effects of two drugs acting at the peripheral type benzodiazepine binding sites, PK 11195 and RO5-4864, were examined in shock-induced suppression of drinking in rats. These two compounds have opposite effects : RO5-4864 (3.1-1205 mg/kg i.p.) enhanced whereas PK 11195 (25-50 mg/kg i.p.) decreased the punished responding, and PK 11195 (6.25 mg/kg, a dose which did not alter the punished responding) blocked the proconflict action of RO5-4864 (6.25 and 12.5 mg/kg). The effects of RO5-4864 and PK 11195 were not antagonized by RO15-1788, a selective antagonist of the central benzodiazepine site. In addition, PK 11195 (6.25 mg/kg) did not reverse the proconflict effect of two beta-carbolines : beta-CEE and FG 7142. AS picrotoxin did not change the punished responding, these data imply that the effects of RO5-4864 and PK 11195 on the one hand and those of chlordiazepoxide and beta-carbolines on the other hand are differentially mediated and suggest that the peripheral type benzodiazepine binding sites are involved in this conflict model.

PK 11195, an antagonist of peripheral type benzodiazepine receptors, modulates Bay K8644 sensitive but not beta- or H2-receptor sensitive voltage operated calcium channels in the guinea pig heart.

In a partially depolarized guinea pig papillary muscle preparation, BAY K8644 stimulated voltage-operated calcium channels, promoting slow action potentials; this effect was dose-dependent over a concentration range of 3 X 10(-7) M to 3 X 10(-6) M. Isoproterenol and histamine also induced slow action potentials by stimulating beta or H2 receptors, respectively. PK 11195, the antagonist of peripheral type benzodiazepine receptors, inhibited the effect of BAY K8644, but not those of histamine or isoproterenol. Moreover, PK 11195 "dose-dependently" antagonized the ability of RO5-4864 to inhibit the slow action potentials elicited by barium chloride. Thus, in the heart, PK 11195, an antagonist of peripheral type benzodiazepine receptors, can modulate voltage-operated calcium channels when they are activated directly, but not when they are activated by stimulation of neurotransmitter receptors.

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.

Peripheral benzodiazepine binding sites: effect of PK 11195, 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide. I. In vitro studies.

[3H] RO5-4864 binding sites have been characterized in kidney, heart, brain, adrenals and platelets in the rat. In all these organs the following order of potency in the RO5-4864 displacement was found: RO5-4864 greater than diazepam greater than clonazepam indicating that they correspond to the "peripheral type" of benzodiazepine binding sites. PK 11195, an isoquinoline carboxamide derivative, displaces [3H] RO5-4864 from its binding sites in all the organs. PK 11195 was as potent as RO5-4864 in the platelets, heart, adrenals, kidney and several brain regions (midbrain, hypothalamus, medulla + pons and hippocampus. However it was 5 to 10 times more effective in cortex and striatum. In conclusion PK 11195 might represent a new tool to elucidate the physiological relevance of "peripheral type" benzodiazepine binding sites and might help to discriminate the hypothetical subclasses of these binding sites.

Peripheral benzodiazepine binding sites: effect of PK 11195, 1-(2-chlorophenyl)-N-methyl-(1-methylpropyl)-3 isoquinolinecarboxamide. II. In vivo studies.

Peripheral type of benzodiazepine binding sites were labelled in the kidney, the heart and the brain with [3H] RO5-4864 following intravenous injection in mice. The regional distribution of this in vivo binding parallels the in vitro binding: heart and kidney were more labelled than brain. Benzodiazepine potencies in reducing [3H] RO5-4864 binding in vivo parallel relative affinities for [3H] RO5-4864 binding sites in isolated organs membranes: RO5-4864 greater than diazepam greater than clonazepam. PK 11195 a new compound, chemically unrelated to benzodiazepines, which is a potent inhibitor of [3H] RO5-4864 in vitro is also very effective (more than RO5-4864) after I.P. injection and oral administration. These results emphasize the feasibility of using this technique to examine the effects on various pharmacological and physiological manipulations of these binding sites in vivo. Moreover the fact that PK 11195 binds to these sites in vivo might indicate that this compound could help to elucidate the physiological relevance of the peripheral type of benzodiazepine binding sites.

Differentiation between two ligands for peripheral benzodiazepine binding sites, [3H]RO5-4864 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.

Opposite effects of an agonist, RO5-4864, and an antagonist, PK 11195, of the peripheral type benzodiazepine binding sites on audiogenic seizures in DBA/2J mice.

Two compounds with high affinity for the "peripheral type" benzodiazepine binding sites, PK 11195 (an isoquinoline derivative) and RO5-4864 (a benzodiazepine derivative) can modify the sensitivity of DBA/2J mice to audiogenic seizures. RO5-4864 (1-15 mg/kg) facilitates in a dose-dependent manner the audiogenic seizures and PK 11195 (2-5 mg/kg) antagonizes the RO5-4864 effects. At these doses PK 11195 alone does not modify the sensitivity to audiogenic seizures, but at doses between 20-80 mg/kg it protects DBA/2J mice against audiogenic seizures. By contrast PK 11195 is inactive against the facilitation of audiogenic seizures by ethyl-beta-carboline-3-carboxylate (a brain benzodiazepine receptor inverse agonist) and against the seizure elicited in absence of noise stimuli by RO5-4864 at doses between 20-40 mg/kg. These results suggest that facilitation by RO5-4864 of the audiogenic seizures and its antagonism by PK 11195 are mediated by the peripheral type benzodiazepine binding sites and agree with the thermodynamic analysis of the binding data which suggested that RO5-4864 might be an agonist and PK 11195 an antagonist. The good correlation between pharmacological effects and the occupancy degree of the binding sites as measured by the displacement of the "in vivo" [3H]-PK 11195 binding give an additional support to binding sites mediated effects.

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.

Histidine modification with diethylpyrocarbonate induces a decrease in the binding of an antagonist, PK 11195, but not of an agonist, RO5-4864, of the peripheral benzodiazepine receptors.

[3H]PK 11195 binding to peripheral type benzodiazepine binding sites in kidney membranes is inhibited by the histidine blocking agent diethylpyrocarbonate. This reagent irreversibly decreases the Bmax for [3H]PK 11195 without affecting the affinity. By contrast binding of [3H]RO5-4864 is not affected by diethylpyrocarbonate treatment. However RO5-4864 can protect in a concentration dependent manner the [3H]PK 11195 binding site from diethylpyrocarbonate whereas clonazepam and RO15-1788 are not active. These results suggest that PK 11195 and RO5-4864 interact with different conformational states of the receptors that RO5-4864. This is in agreement with our previous hypothesis that PK 11195 is an antagonist and RO5-4864 an agonist at the "peripheral type" benzodiazepine receptors.

Modulation of voltage-operated, but not receptor-operated, calcium channels in the rabbit aorta by PK 11195, an antagonist of peripheral-type benzodiazepine receptors.

We compared the effects of PK 11195, an antagonist of peripheral benzodiazepine receptors, on contractions of rabbit aorta by activation of either voltage-operated calcium channels (VOC) using BAY K 8644 (a calcium "agonist") and KCl or receptor-operated channels (ROC) using phenylephrine and B-HT 920, (alpha 1- and alpha 2-adrenoceptor agonist, respectively). In partially depolarized muscle strips, BAY K 8644 induced contractions that were noncompetitively inhibited by PK 11195 (pD'2 = 5.6 +/- 0.15). The effect of this calcium agonist was also antagonized by nitrendipine (competitively) and by yohimbine (noncompetitively), while prazosin was inactive. Contractions induced by KCl were inhibited by nitrendipine and, weakly, by PK 11195. Contractions induced by phenylephrine and B-HT 920 were inhibited competitively by prazosin and yohimbine and noncompetitively by nitrendipine, while PK 11195 was ineffective. It is concluded that PK 11195 behaves as an antagonist of VOC activated by BAY K 8644 and to a lesser extent by KCl depolarization but not of ROC coupled to alpha 1- and alpha 2-receptors.

Pharmacology of peripheral type benzodiazepine receptors in the heart.

RO5-4864 decreased in a dose-dependent manner the duration of intra cellular action potential and the contractility in the guinea pig papillary muscle. Diazepam was less active and clonazepam inactive. The effects of RO5-4864 were blocked by PK 11195 but not by RO15-1788. These results indicate that peripheral type benzodiazepine binding sites are pharmacological receptors. PK 11195 antagonized the increase and the decrease in the duration of intracellular action potential induced by BAY K-8644 and calcium channel blockers (nitrendipine, diltiazem, verapamil) respectively but not the increase induced by tetraethylammonium which blocks potassium channel. Moreover the decrease in contractility provoked by RO5-4864 was antagonized by 4 mM Ca2+. Thus peripheral type BZ receptors are coupled with Ca2+ channels in the heart.