The subcellular location in rat kidney of the peripheral benzodiazepine acceptor

Abstract

In rat kidney high-affinity binding sites for [3H]Ro-5-4864 and [3H]PK-11195 with the properties of the peripheral-type acceptor were found enriched in mitochondrial (M) and light-mitochondrial-lysosomal (L) fractions on differential centrifugation. When the combined M and L fractions were subjected to sucrose density gradient centrifugation, these binding sites were found enriched at a density of 1.155 g/ml coincident with a population of light mitochondria, whereas a population of heavier mitochondria ( Q = 1.175 g/ml) had few or no binding sites. Transmission electron microscopy showed that whereas the heavier mitochondria appeared highly pure and intact, the lighter mitochondria appeared less intact and to be contaminated with vesicular structures. After fractionation of the light mitochondria and vesicles by centrifugation, both fractions showed the same ratio of [3H]Ro-4864 binding sites to monoamine oxidase activity consistent with the vesicles being of mitochondrial outer-membrane origin.Digitonin pre-treatment had no effect on the density of acceptor-rich fractions on sucrose density gradient centrifugation. However, pretreatment with succinate/iodophenylnitrophenylphenyltetrazolium (INT) perturbed equally the density of acceptor-rich fractions and mitochondrial marker enzymes.When mitochondrial fractions were subjected to sonication prior to density gradient centrifugation the binding sites were now found highly enriched in a much lighter fraction coincident with the monoamine oxidase activity and thus consistent with being outer-membrane vesicles.When a mitochondrial fraction was subjected to hypotonic treatment before assay no evidence for activation/ unmasking of binding sites was found. The hypotonic treatment did not release any inhibitor of the binding sites.These results are consistent with the peripheral benzodiazepine acceptor having an outer-membrane location on a sub-population of rat kidney mitochondria. Those mitochondria showing high levels of the acceptor are either light mitochondria or appear more susceptible to osmotic damage than those mitochondria in which the acceptor is absent or at low levels.The benzodiazepines have anxiolytic, sedative and anticonvulsant actions probably mediated through allosteric modulation of neuronal post-synaptic plasma membrane 4-aminobutyrate-gated chloride channels [l , 21. Whilst the structural analysis of these channel proteins is well advanced [3], the properties and function of a second-class of benzodiazepine-binding sites are still unclear. These nonneuronal or peripheral-type benzodiazepine acceptors [4] appear not to be linked to 4-aminobutyrate-gated chloride channels [5,6], show a different binding profile for benzodiazepines than for the 4-aminobutyrate receptor [4-61 and appear to have different polypeptide subunit M , values depending on the nature of photoaffinity labelling reagents [7, 81. Evidence from various laboratories has suggested that benzodiazepines showing preference for the peripheral-type

Localization of the peripheral-type benzodiazepine binding site to mitochondria of human glioma cells

“…Various groups have suggested that PBBS are localized to mitochondria [17,18], plasma membrane [19,22] or nuclei [5,21,22]. The enrichment of markers for these components was compared to the enrichment of [3H]PK 11195 binding in each of six subcellular fractions (Fig.…”

“…PBBS ligands have been shown to influence mitochondrial respiration in rat kidney [16] and PBBS have been localized to mitochondria in rat kidney [17] and rat adrenal [18]. Binding sites with nearly identical drug binding parameters however, have been localized to human erythrocyte membrane preparations [19] which lack mitochondria and nuclei [20].…”

Effects of Vinpocetine on mitochondrial function and neuroprotection in primary cortical neurons

“…In the last decade, the involvement of PBRs in neurological conditions was extensively investigated using PBR ligands or PBR overexpressing cells (Gavish et al, 1999;Fischer et al, 2001;Casellas et al, 2002;Papadopoulos et al, 2006b). Accordingly, PBRs were found to be preferentially localised in the outer membrane of mitochondria (O'Beirne and Williams, 1988;Hirsch et al, 1989). Several studies indicated that PBR-mediated changes in mitochondrial function can play a key role during apoptotic events (Tanimoto et al, 1999;Casellas et al, 2002;Chelli et al, 2004;Azarashvili et al, 2005;Li et al, 2007).…”

Les endozépines, facteurs locaux de régulation de la stéroïdogenèse testiculaire

“…Des exp6riences de liaison du PK11195, ligand sp6cifique des PBR, men6es dans la surr6nale, le testicule, le poumon, le rein, le cceur, le muscle squelettique, le foie et le cerveau du rat ont montr6 que les PBR sont localis6s au niveau de la membrane mitochondriale [5,6,14,95,96]. Des 6tudes compl6mentaires r6alis6es dans la surr6nale [5], le rein et le foie [95,96] ainsi que dans la cellule de Leydig [103] ont permis de pr6ciser la localisation des PBR sur la membrane mitochondriale externe.…”

“…Des 6tudes compl6mentaires r6alis6es dans la surr6nale [5], le rein et le foie [95,96] ainsi que dans la cellule de Leydig [103] ont permis de pr6ciser la localisation des PBR sur la membrane mitochondriale externe. Cependant, la pr6sence de PBR sur la membrane mit0chondriale interne a 6galement 6t6 rapport6e dans le poumon du cobaye [92] ce qui sugg~-re que ces r6cepteurs pourraient 8tre localis6s au niveau des sites de contact entre les membranes mitochondria!es internes et externes [103].…”