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.     

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.     

Comparison of two PBR ligands with classical antiinflammatory drugs in LPS-induced arthritis in rats

The antinociceptive and anti-edematogenic effects of peripheral benzodiazepine receptor (PBR) ligands, Ro5-4864 (7-chloro-5- (4-chlorophenyl)-1,3-dihydro-1-methyl-2-H-1,4-benzodiazepine-2) and PK11195 (1-(2-chlorophenyl)-N-methyl-N(1-methylpropyl)-3-isoquinoline carboxamide), were studied in an experimental model of carrageenan/LPS -induced arthritis in rats. These effects were compared with those of indomethacin and dexamethasone. Both pre and post-treatments with PK11195 were found to be anti-edematogenic and antinociceptive. The lower dose (0.01 mg/kg) exhibited the higher anti-edematogenic effect. On the other hand, the higher dose (0.5 mg/kg) produced antinociception, but with a decreased anti-edematogenic effect. Ro5-4864 produced a negligible antinociception and anti-edematogenic effect as pretreatment, but a pro-edematogenic effect when given as post-treatment. Dexamethasone and indomethacin presented parallel and dose-dependent antinociceptive and anti-edematogenic effects. In conclusion, PK11195 can effectively diminish arthritic nociception and edema elicited by LPS, but probably by mechanisms different from those of dexamethasone or indomethacin. RO5-4864 seemed to have opposite effect on this model.     

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.     

The Peripheral-Type Benzodiazepine Receptor Ligands [3H]Ro 5-4864 and [3H]PK 11195 Bind to the Retina of Rabbit, but Not of Turtle

Abstract: The binding of [³H]flunitrazepam, [³H]RO 5-4864, and [³H]PK 11195 to membrane preparations of the retina was studied in the turtle and rabbit. Only a single population of [³H]flunitrazepam binding sites was detected in the turtle, whereas two populations appeared to be present in the rabbit. No specific binding for [³H]RO 5-4864 and [³H]PK 11195 could be detected in the turtle. In rabbit, both ligands bound with high affinity, revealing a significant population of binding sites (K_D values of 24 ± 2.3 and 2.2 ± 0.8 nM, and B_max values of 440 ± 35 and 1,482 ± 110 fmol/mg of protein, respectively). The binding was temperature - and protein-dependent. Displacement studies showed a similar rank order of potency of various unlabeled ligands against both [³H]RO 5-4864 and [³H]PK 11195 (PK 11195 > Ro 5-4864 > flunitrazepam > flumazenil). These results suggest that peripheral-type benzodiazepine receptors are present in the retina of the rabbit, but not of the turtle.     

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.     

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.     

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.     

Ontogeny of susceptibility to the convulsant, Ro 5-4864, and its relationship to audiogenic seizure susceptibility in inbred mice

The postnatal development of susceptibility to the convulsant effects of Ro5-4864 (4'-chlorodiazepam) was characterized in two inbred mouse strains (DBA/2J and BALB/c ByJ) which as adults differ markedly in their response to this convulsant. Onset of susceptibility to a dose of Ro5-4864 which caused a high frequency of clonic seizures in adults was observed at 10 days of age in DBA/2 mice, but not until 35 days in BALB/c By mice. At 14 days of age an abrupt increase in susceptibility to Ro5-4864-induced tonic seizures was found in DBA/2 but not BALB/c By mice. Both the peak of tonic seizure susceptibility (21 days) and the time course of its subsequent age-dependent decline closely paralleled the change in audiogenic seizure susceptibility in the DBA/2 strain. PK11195 (40 mg/kg) blocked Ro5-4864 (25 mg/kg)-induced, age-dependent tonic seizures but had no effect on clonic seizure induction in the same mice. These observations establish that both the susceptibility to Ro5-4864 in adult mice and the postnatal time course for development of susceptibility to this convulsant are inherently different in these two strains of mice. The lack of coincidence between the developmental onset of susceptibility to Ro5-4864-induced seizures and the onset of supersensitivity to Ro5-4864-induced tonic seizures during the period of peak audiogenic seizure susceptibility in DBA/2 mice implies that more than one neurochemical mechanism is involved in the ability of Ro5-4864 to induce seizures in this strain. However, the blockade of Ro5-4864-induced tonic seizures by PK11195 suggests that peripheral type benzodiazepine receptors may mediate this effect.     

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.     

Peripheral-type benzodiazepine receptors in human cerebral cortex, kidney, and colon.

The specific binding of [3H]PK 11195 and [3H]Ro 5-4864 to human cerebral cortex, kidney, and colon membranes was studied in order to determine whether peripheral type benzodiazepine receptors (PBR) characteristics located in human tissues are similar to those located in calf or rat tissues. While [3H]PK 11195 (0.05-10 nM, final concentration) bound with high affinity (KD about 2 nM) to human cerebral cortex, kidney, and colon membranes, yielding maximal numbers of binding sites of 255 +/- 23, 1908 +/- 28, and 1633 +/- 98 fmol/mg protein, respectively, the specific binding of [3H]Ro 5-4864 (1.25-40 nM, final concentration), was barely detectable (nonspecific binding about 90% of the total binding). Furthermore, unlabeled PK 11195 was two orders of magnitude more potent than unlabeled Ro 5-4864 in displacing [3H]PK 11195 specific binding from human cerebral cortex and kidney membranes. These results indicate that PBR binding characteristics located in human tissues are similar (but not identical) to those located in calf tissues, but not to those located in rat tissues.     

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.     

Involvement of steroids in anti-inflammatory effects of peripheral benzodiazepine receptor ligands

Mouse paw oedema induced by carrageenan is used to determine if glucocorticoids are involved in the anti-inflammatory effects of peripheral benzodiazepine receptor ligands. The anti-inflammatory responses elicited by i.p. treatment with 1-(2-chlorophenyl)-N-methyl-N (1-methyl-propyl)-3-isoquinoline carboxamide (PK11195) and 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2-H-1, 4-benzodiazepin-2 (Ro5-4864) were reversed by aminoglutethimide, an inhibitor of steroidal synthesis. Intraplantar injection into the ipsilateral paw of Ro5-4864, but not PK11195, inhibited the formation of paw oedema and this effect was reversed by aminoglutethimide. These results suggest that glucocorticoids are involved in the systemic and local anti-inflammatory effects of Ro5-4864 and only in the systemic response to PK11195.     

Do benzodiazepine receptors mediate the anticonflict action of pentobarbital?

The effects of the benzodiazepine antagonist CGS 8216 (2-phenylpyrazolo[4,3-c]quinoline-3(5H)-one) were examined in a thirsty rat conflict test in the presence and absence of pentobarbital. CGS 8216 (2.5-10 mg/kg i.p.) did not affect nonpunished responding, but doses of 5 and 10 mg/kg significantly reduced the rate of punished responding (i.e., the number of 3 second drinking episodes in a "shock" contingency). However, a dose of CGS 8216 which did not significantly alter punished responding (2.5 mg/kg) antagonized the anticonflict actions of pentobarbital. These observations suggest that while high doses of CGS 8216 may elicit an "anxiogenic" response in rodents, lower doses of CGS 8216 antagonize the anticonflict actions of a compound which has been shown to enhance benzodiazepine affinity in vitro. These data imply that the anticonflict actions of pentobarbital may be mediated through benzodiazepine receptors.     

High-affinity binding sites for PK 11195, but not for RO5-4864, in porcine aortic smooth muscle

Peripheral benzodiazepine (BZ) binding sites were characterized in porcine aortic smooth muscle membrane preparation. [3H]PK11195 bound with high affinity to the membranes (Kd = 8.6 + 0.9 nM), whereas [3H]Ro5-4864 bound slightly to the membranes. The Ki value of Ro5-4864 obtained from the inhibition of [3H]PK 11195 binding was 1200 + 200 nM, which was 480 times weaker than that obtained in rat kidney. Furthermore, the Ro5-4864 effect was temperature-insensitive. When [3H]PK 11195 binding was examined in porcine, human and rat platelets, Ro5-4864 inhibited the binding in porcine and human platelets one order of magnitude less potently than that in rat platelets. These results suggest that low affinity for Ro5-4864 in porcine aorta smooth muscle originates in porcine tissue, but not in smooth muscle.     

Presence of a peripheral type benzodiazepine binding site on the macrophage; its possible role in immunomodulation

Saturable binding site for 3H-flunitrazepam (KD = 43 +/- 7 nM, Bmax = 391 +/- 58 fmoles/cell, i.e. 250,000 sites/cell) is characterized on Mouse peritoneal inflammatory macrophages. The affinity for different ligands (PK 11195 greater than Ro 5-4864 greater than diazepam greater than flunitrazepam greater than clonazepam greater than Ro 15-1788) shows that this site is of peripheral type. In vivo the humoral response in Mice to Sheep red blood cells was stimulated by administration of 1 mg/kg of PK 11195 (+85%), Ro 5-4864 (+80%) and diazepam (+58%). Clonazepam and Ro 15-1788 are devoid of activity. This suggests that molecules which show affinity for the "peripheral type" benzodiazepine binding site might modulate the immune response.     

Behavioral effects and benzodiazepine antagonist activity of Ro 15-1788 (flumazepil) in pigeons

The selective benzodiazepine receptor antagonist, Ro 15-1788, produced behavioral effects in pigeons at doses at least 100 times lower than those previously reported to possess intrinsic pharmacological activity in mammals. In contrast to its effects in mammalian species, in pigeons, Ro 15-1788 does not exhibit partial agonist activity. Key-peck responses of pigeons were studied under a multiple fixed-interval 3-min, fixed-interval 3-min schedule in which the first response after 3-min produced food in the presence of red or white keylights. In addition, every 30th response during the red keylight produced a brief electric shock (punishment). Under control conditions, punished responding was suppressed to 30% of unpunished response levels. Ro 15-1788 (0.01 mg/kg, i.m.) increased unpunished response rates by 33% without affecting rates of punished responding. Doses of 0.1 to 1.0 mg/kg Ro 15-1788 produced dose-related decreases in both punished and unpunished responding. As is characteristic of other benzodiazepines, midazolam (0.1 and 0.3 mg/kg, i.m.) markedly increased punished responding but had little effect on rates of unpunished responding. Ro 15-1788 antagonized the increases in punished responding and also reversed the rate-decreasing effects of higher doses of midazolam. However, the effectiveness of Ro 15-1788 as a benzodiazepine antagonist was limited by its intrinsic activity: rate-decreasing doses of Ro 15-1788 were unable to completely reverse behavioral effects of midazolam. Midazolam was an effective antagonist of the behavioral effects of Ro 15-1788 (up to 0.1 mg/kg) but midazolam did not influence the rate-decreasing effects of 1.0 mg/kg Ro 15-1788 across a 100-fold dose range. In the pigeon, the behavioral effects of relatively low doses of Ro 15-1788 (0.01-0.1 mg/kg) appear to be related to benzodiazepine receptor mechanisms, whereas other systems appear to be involved in the effects of higher doses.     

Selective pharmacological modulation of renal peripheral-type benzodiazepine binding by treatment with diuretic drugs

We have assessed the effects of in vivo administration of different classes of diuretic drugs on the expression of the peripheral-type benzodiazepine binding site (PBBS) in crude membranes derived from the cortex and outer medulla of rat kidney by saturation analysis with the PBBS-selective ligands [3H]RO5-4864 and [3H]PK 11195 in cortex and [3H]RO5-4864 in outer medulla. Administration for 14-15 days of furosemide, a drug that blocks NaCl-KCl coupled transport in the thick ascending limb of the loop of Henle, produced a significant doubling in the PBBS density (Bmax) in outer medulla, a region of the kidney rich in thick ascending limbs, and produced a lesser but significant increase in PBBS density in the cortex. Conversely, administration for 14-15 days of the carbonic anhydrase inhibitor acetazolamide, which acts predominantly in the proximal tubule, and hydrochlorothiazide, which acts predominantly in the early distal tubule, elicited statistically significant increases in PBBS density in renal cortex but not in renal outer medulla. Furthermore, all drug treatments were without effect on the equilibrium dissociation constants (Kds) of [3H]RO5-4864 and [3H]PK 11195 binding to cortical and outer medullary membrane preparations. These findings demonstrate that the PBBS can be selectively "up-regulated" in different regions of the kidney by diuretic drugs with different modes/sites of action.     

Isolation and Characterization of a Rat Brain Triakontatetraneuropeptide, a Posttranslational Product of Diazepam Binding Inhibitor: Specific Action at the Ro 5-4864 Recognition Site

This report describes the purification and characterization from rat brain of triakontatetraneuropeptide (TTN, DBI 17-50), a major biologically active processing product of diazepam binding inhibitor (DBI). Brain TTN was purified by immunoaffinity chromatography with polyclonal octadecaneuropeptide, DBI 33-50) antibodies coupled to CNBr-Sepharose 4B followed by two reverse-phase HPLC steps. The amino acid sequence of the purified peptide is: Thr-Gln-Pro-Thr-Asp-Glu-Glu-Met-Leu-Phe-Ile-Tyr-Ser-His-Phe-Lys-Gln-Ala-Thr-Val - Gly-Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Leu-Leu-Asp-Leu-Lys. Synthetic TTN injected intracerebroventricularly into rats induces a proconflict activity (IC50 0.8 nmol/rat) that is prevented by the specific "peripheral" benzodiazepine (BZ) receptor antagonist isoquinoline carboxamide, PK 11195, but not by the "central" BZ receptor antagonist imidazobenzodiazepine, flumazenil. TTN displaces [3H]Ro 5-4864 from synaptic membranes of olfactory bulb with a Ki of approximately 5 microM. TTN also enhances picrotoxinin inhibition of gamma-aminobutyric acid (GABA)-stimulated [3H]flunitrazepam binding. These data suggest that TTN, a natural DBI processing product acting at "Ro 5-4864 preferring" BZ binding site subtypes, might function as a putative neuromodulator of specific GABAA receptor-mediated effects.     

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.