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

[³H] R05-4864 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 R05-4864 displacement was found : R05-4864 > diazepam > clonazepam indicating that they correspond to the “peripheral type” of benzodiazepine binding sites. PK 11195, an isoquinoline carboxamide derivative, displaces [³H] R05-4864 from its binding sites in all the organs. PK 11195 was as potent as R05-4864 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.     

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

R05-4864 decreased in a dose-dependent manner, from 3 × 10^?9 M to 3 × 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 R05-4864 were GABA-independent and antagonized by PK 11195 but not by the selective antagonist of the brain type benzodiazepine receptors R015-1788. These results show the pharmacological relevance of peripheral type benzodiazepine binding sites at the cardiac level.     

Benzodiazepine receptor-dependent modulation of neutrophil (PMN) free amino- and alpha-keto acid profiles or immune functions

Summary. We have examined the effects of midazolam, Ro 5-4864 (agonist for peripheral [p] benzodiazepine receptors [BR]), PK 11195 (antagonist for pBR), flumazenil (antagonist for central BR), naloxone (antagonist for opiate receptors) and the combination of midazolam and Ro 5-4864, PK 11195, flumazenil or naloxone on intracellular amino- and -keto acids and the immune function markers superoxide anion (O₂^–), hydrogen peroxide (H₂O₂) and released myeloperoxidase (MPO) activity in neutrophils (PMN). Only midazolam and Ro 5-4864 led to significant changes in the dynamic PMN free amino- and -keto acid pools. Concerning PMN immune function markers, midazolam and Ro 5-4864 significantly decreased O₂^– and H₂O₂ formation and released MPO. When midazolam and Ro 5-4864 were applied together they appeared to act additively. Pre-incubation with PK 11195 partially neutralized the midazolam effects whereas flumazenil or naloxone showed no effects. We therefore believe that pBR are involved in the signal transmission of anesthetic-induced cellular metabolic changes in PMN.     

Effect of peripheral benzodiazepine receptor (PBR/TSPO) ligands on opening of Ca<Superscript>2+</Superscript>-induced pore and phosphorylation of 3.5-kDa polypeptide in rat brain mitochondria

The effect of nanomolar concentrations of PBR/TSPO ligands—Ro 5-4864, PK11195, and PPIX—on Ca²⁺-induced permeability transition pore (PTP) opening in isolated rat brain mitochondria was investigated. PBR/TSPO agonist Ro 5-4864 (100 nM) and endogenous ligand PPIX (1 μM) were shown to stimulate PTP opening, while antagonist PK11195 (100 nM) suppressed this process. Correlation between PBR ligand action on PTP opening and phosphorylation of a 3.5 kDa polypeptide was investigated. In intact brain mitochondria, incorporation of [γ-³²P]ATP into 3.5 kDa peptide was decreased in the presence of Ro 5-4864 and PPIX and increased in the presence of PK11195. At threshold Ca²⁺ concentrations leading to PTP opening, PBR/TSPO ligands were found to stimulate dephosphorylation of the 3.5 kDa peptide. Specific anti-PBR/TSPO antibody prevented both PTP opening and dephosphorylation of the 3.5-kDa peptide. The peptide was identified as subunit c of F_oF₁-ATPase by Western blot using specific anti-subunit c antibody. The results suggest that subunit c of F_oF₁-ATPase could be an additional target for PBR/TSPO ligands action, is subjected to Ca²⁺- and TSPO-dependent phosphorylation/dephosphorylation, and is involved in PTP operation in mitochondria.     

Partial Purification and Pharmacology of Peripheral-Type Benzodiazepine Receptors

Abstract

This report describes the results obtained with a new photo-affinity ligand for the “peripheral-type” benzodiazepine binding site (PBS), using a digitonin solubilized preparation from rat heart or adrenals.

The specific binding activity of the solubilized adrenal preparation is higher than 50 pmo1/mg protein, with binding proper-ties and pharmacological specificity identical to the membrane bound PBS. The apparent molecular weight of the solubilized PBS, determined by gel filtration is 215 KDa.

The photoaffinity ligand (PK 14105) is a nitrophenyl derivative of PK 11195, which attaches covalently and specifically to all the PBS when cardiac membranes are irradiated with this compound under ultraviolet light. After photolabelling with [³H]PK 14105 and solubilization in SDS of heart or adrenal membranes, gel electrophoresis indicates the existence of a single protein band whose molecular weight (18 KDa) is unaltered by incubation with sulphydryl-reducing or protein cross-linking agents. This molecule seems to be a low molecular weight, acidic protein.

Diethylpyrocarbonate decreases partially (60 %) the binding of [³H]PK 11195 without affecting [³H] RO5-4864 binding, which implies a vital histidine residue in the binding domain of [³H] -PK 11195. Treatment with phospholipase A₂ or mellitin, a stimulant of endogenous PLA₂, led to a selective, loss of [³H]RO5-4864 binding with no change in the binding of [³H]PK 11195.

Such differences between a benzodiazepine ligand and an isoquinoline ligand suggest that these compounds may induce.     

The bovine peripheral-type benzodiazepine receptor: a receptor with low affinity for benzodiazepines.

The density of bovine peripheral-type benzodiazepine receptors (PBR) in four tissues was highest in adrenal cortex. The adrenal cortex PBR cofractionated with a mitochondrial membrane marker enzyme and could be solubilized with intact ligand binding properties using digitonin. The membrane bound and soluble mitochondrial receptors were pharmacologically characterized and showed the rank order of potency to inhibit [3H]PK 11195 binding was PK 11195 greater than protoporphyrin IX greater than benzodiazepines (clonazepam, diazepam, or Ro5-4864). [3H]PK 11195 binding to bovine adrenal mitochondria was unaffected by diethylpyrocarbonate, a histidine residue modifying reagent that decreased binding to rat liver mitochondria by 70%. [3H]PK 14105 photolabeled the bovine PBR and the Mr was estimated under nondenaturing (200 kDa) and denaturing (17 kDa) conditions. These results demonstrate the bovine peripheral-type benzodiazepine receptor is pharmacologically and biochemically distinct from the rat receptor, but the receptor component photolabeled by an isoquinoline ligand has a similar molecular weight.     

Effect of PK11195, a peripheral benzodiazepine receptor agonist,on insulinoma cell death and insulin secretion

Functional role of peripheral benzodiazepine receptor on mitochondrial membrane in apoptosis and insulin secretion from insulinoma cells was studied. A prototypic peripheral benzodiazepine receptor agonist PK11195 induced insulinoma cell apoptosis, while a central benzodiazepine receptor agonist did not. Death of insulinoma cells by PK11195 was inhibited by cyclosporin A,{ a blocker of mitochondrial permeability transition pore}. Caspase inhibitors further inhibited MIN6N8 cell death. PK11195 induced dissipation of mitochondrial potential and cytochrome c translocation to cytoplasm. PK11195 induced an increase in cytoplasmic [Ca^{2 +}], which was reversed by cyclosporin A. Rhod-2 staining showed decreased mitochondrial [Ca^{2 +}] after PK11195 treatment. PK11195 potentiated glucose-induced insulin secretion probably due to the increased cytoplasmic [Ca^{2 +}]. Calpain was activated following Ca^{2 +} release, and calpain inhibitors attenuated death of insulinoma cells by PK11195. These results suggest that PK11195 induces mitochondrial potential loss, cytochrome c translocation, increased insulin secretion in conjunction with an increase in cytoplasmic [Ca^{2 +}] and calpain activation, which collectively leads to apoptosis of insulinoma cells.     

Effect of Ro 5-4864 and PK11195 on protein phosphorylation in mitochondria isolated from primary cultures of rat astrocytes

Astrocytes are the most numerous cell type within the central nervous system. Earlier, high-affinity binding sites for [³H]PK 11195 and [³H]Ro 5-4864 with the properties of the peripheral-type benzodiazepine receptor were detected in primary cultures of astrocytes. TSPO/PBR was shown to be localized in mitochondria. Recently, we showed that TSPO/PBR ligands, Ro 5-4864 and PK11195, were able to modulate the function of non-specific pore (PTP) in brain and liver mitochondria as well as protein phosphorylation in the presence of threshold calcium concentrations. In the present study for the first time the function of astrocyte mitochondria were studied under condition of PTP opening. Parameters of PTP induction were measured by means of simultaneous registrations of the membrane potential, calcium accumulation and calcium release as well as detection of the oxygen consumption with selective electrodes. Four phosphorylated proteins in range of 67 kDa, 46 kDa, 48 kDa and 3.5 kDa have been found under these conditions. It was established that in astrocyte mitochondria TSPO/PBR exists in monomer form (18 kDa). The phosphorylation level of these proteins was found to be modulated by TSPO/PBR ligands, Ro 5-4864 and PK11195, in a range of concentrations from 0.01 to 1 μM, in the same way as it was earlier described for brain mitochondria [Azarashvili et al., J Neurochem., 2005].     

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

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 congruent to 10(-9) M) [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(-9) M). 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 degrees C (PK 11195 greater than RO5-4864 greater than diazepam greater than dipyridamole greater than 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.     

Heterogeneity Between Rat and Calf Peripheral-Type Benzodiazepine Binding Sites: Differential Sensitivity to Triton X-100

Abstract

The effect of various detergents treatment on the specific binding of [³H]PK 11195 (2nM) to peripheral-type benzodiazepine binding sites (PBS) in calf and rat kidney, adrenal gland, and cerebral cortex membranes was studied. At a concentration of 0.025%, Triton X-100 increased [³H]PK 11195 specific binding to calf kidney, adrenal gland, and cerebral cortex membranes by 20–40%. At the same concentration, Triton X-100 scarcely affected specific binding of [³H]PK 11195 to rat cerebral cortex but decreased binding to rat kidney and adranal gland membranes by 20–30%. At a concentration of 0.05% of Triton X-100, [³H]PK 11195 specific binding to calf kidney, adrenal gland, and cerebral cortex membranes was increased by 10–20%; whereas [³H]PK 11195 specific binding to rat kidney, adrenal gland, and cerebral cortex membranes was decreased by more than 40%. The increase in [³H]PK 11195 specific binding to calf kidney membranes following Triton X-100 (0.05%) treatment was apparently due to an increase in the binding affinity of PBS, since the density remained unaltered; whereas, the decrease in [³H]PK 11195 specific binding to rat kidney membranes was due to a decrease in both binding affinity and density of PBS. On the other hand, the detergents 3- [(3- cholamidopropyl)- dimethylammonio] - 1 - propane sulfonate (CHAPS), Tween 20, deoxycholic acid, and digitonin have a similar effect on [³H]PK 11195 specific binding to PBS in both calf and rat kidney membranes.     

The ACh-evoked,Ca2+-activated Whole-cell K+ Current in Mouse Mandibular Secretory Cells. Whole-cell and Fluorescence Studies

In our previous studies on sheep parotid secretory cells, we showed that the K⁺ current evoked by acetylcholine (ACh) was not carried by the high-conductance voltage- and Ca²⁺-activated K⁺ (BK) channel which is so conspicuous in unstimulated cells, notwithstanding that the BK channel is activated by ACh. Since several studies from other laboratories had suggested that the BK channel did carry the ACh-evoked K⁺ current in the secretory cells of the mouse mandibular gland, and that the current could be blocked with tetraethylammonium (TEA), a known blocker of BK channels, we decided to investigate the ACh-evoked K⁺ current in mouse cells more closely. We studied whether the ACh-evoked K⁺ current in the mouse is inhibited by TEA and quinine. Using the whole-cell patch-clamp technique and microspectrofluorimetric measurement of intracellular Ca²⁺, we found that TEA and quinine do inhibit the ACh-evoked K⁺ current but that the effect is due to inhibition of the increase in intracellular Ca²⁺ evoked by ACh, not to blockade of a K⁺ conductance. Furthermore, we found that the K⁺ conductance activated when ionomycin is used to increase intracellular free Ca²⁺ was inhibited only by quinine and not by TEA. We conclude that the ACh-evoked K⁺ current in mouse mandibular cells does not have the blocker sensitivity pattern that would be expected if it were being carried by the high-conductance, voltage- and Ca²⁺-activated K⁺ (BK) channel. The properties of this current are, however, consistent with those of a 40 pS K⁺ channel that we have reported to be activated by ACh in these cells [16]. Received: 9 January 1996/Revised: 17 April 1996     

Effects of W-7 on catecholamine release and 45Ca2+ uptake in cultured adrenal chromaffin cells.

Effects of N-(6-aminohexyl)-5-chloro-1-naph-thalenesulfonamide (W-7), a calmodulin antagonist, on catecholamine (CA) release and ⁴⁵Ca²⁺ uptake were studied using cultured bovine adrenal chromaffin cells. W-7 inhibited the carbamylcholine (CCh)-evoked CA release and ⁴⁵Ca²⁺ uptake in a concentration-dependent manner. The inhibitory effect of W-7 on CCh-evoked CA release was not overcome either by an increase in extracellular calcium or CCh concentration. Although W-7 inhibited the high K⁺-evoked CA release and ⁴⁵Ca²⁺ uptake, potency of the drug was approximately 50–100 fold less than when inhibiting the CCh-evoked CA release and ⁴⁵Ca²⁺ uptake. The inhibitory effects of W-7 were observed both in norepinephrine release and epinephrine release. Moreover, W-7 inhibited the CCh-evoked ⁴⁵Ca²⁺ efflux. These results suggest that the inhibition of CA release by W-7 in adrenal chromaffin cells is mainly due to its inhibition of calcium uptake. W-7 may influence the linkage between acetylcholine-receptor and calcium uptake with higher potency than depolarization-dependent calcium entry.     

Interactions between peripheral-type benzodiazepine receptor ligands and an activator of voltage-operated calcium channels.

The effects of the peripheral-type benzodiapine receptor (PBR) ligands Ro 5-4864 and PK 11195 were studied in the spontaneously beating guinea pig atrium and in a model for myocardial ischemia in the rat. In the former, Bay K 8644 produced positive chronotropic and inotropic responses; intracarotid administration of this agonist (5 or 10 micrograms kg-1) to anesthetized rats elicited a transient increase in mean arterial blood pressure accompanied by alterations in the ECG pattern. Ro 5-4864 and PK 11195 (10 microM) completely blocked the positive chronotropic effect of Bay K 8644 in the atrium, PK 11209, a structural analog of PK 11195 with a low affinity for PBR, was inactive, and the central benzodiazepine receptor ligand clonazepam had a marginal effect. Ro 5-4864 potentiated whereas PK 11195 inhibited the myocardial ischemia produced by Bay K 8644 in the rat. Furthermore, PK 11195 blocked the combined response to Bay K 8644 and Ro 5-4864. Addition of Ro 5-4864 (10 microM) to the organ bath potentiated the inotropic effect of Bay K 8644 in the atria; PK 11195 at the same concentration inhibited this effect. Clonazepam and PK 11209 were both inactive in this regard. Nifedipine, a potent calcium channel antagonist, completely blocked the inotropic and chronotropic responses to Bay K 8644. PK 11195 and Ro 5-4864 did not affect this action. These findings strongly suggest that there is a functional association between PBR and voltage-operated calcium channels in the guinea pig atrium and rat cardiovascular system.     

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.     

Evidence for diabetes-induced alterations in the sulfation of heparin sulfate intestinal epithelial cells

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.     

Behavioural actions of Ro 5-4864: A peripheral-type benzodiazepine?

Ro 5-4864 is a 1,4 benzodiazepine lacking typical benzodiazepine behavioural actions, and which has very low affinity for the “classical” CNS benzodiazepine binding sites. However, Ro 5-4864 has very high affinity for the peripheral type of binding site in the periphery and in the brain. Evidence is reviewed that Ro 5-4864 is sedative, convulsant and anxiogenic in rodents. We also describe the effects of combining Ro 5-4864 treatment with benzodiazepines (e.g. diazepam, chlordiazepoxide) and with other drugs that modify the activity of benzodiazepines (Ro 15-1788, CGS 8216, picrotoxin, PK 11195, phenytoin). The binding sites that might be mediating these behavioural actions of Ro 5-4864 are discussed.     

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.     

Coexistence of central and peripheral benzodiazepine binding sites in the human pineal gland

The pineal gland and particularly its major hormone, melatonin, may participate in several physiological functions, including sleep promotion, anticonvulsant activity and the modulation of biological rhythms and affective disorders. These effects may be related to an interaction with benzodiazepine receptors, which have been demonstrated to be present in the pineal gland of several species including man. The present study examined the characteristics of benzodiazepine binding site subtypes in the human pineal gland, using [³H] flunitrazepam and [³H] PK 11195 as specific ligands for central and peripheral type benzodiazepine binding sites respectively. Scatchard analysis of [³H] flunitrazepam binding to pineal membrane preparations was linear, indicating the presence of a single population of sites. Clonazepam and RO 15-1788, which have a high affinity for central benzodiazepine binding sites, were potent competitors for [³H] flunitrazepam binding in the human pineal, whereas RO 5-4864 had a low affinity for these sites. Analyses of [³H] PK 11195 binding to pineal membranes also revealed the presence of a single population of sites. RO 5-4864, a specific ligand for peripheral benzodiazepine binding sites was the most potent of the drugs tested in displacing [³H] PK 11195, whereas clonazepam and RO 15-1788 were weak inhibitors of [³H] PK 11195 binding to pineal membranes. Overall, these results demonstrate, for the first time, the coexistence of peripheral and central benzodiazepine binding sites in the human pineal gland.     

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.