Moshe Gavish

Channel-like functions of the 18-kDa translocator protein (TSPO): regulation of apoptosis and steroidogenesis as part of the host-defense response.

Due to its channel-like properties, the peripheral-type benzodiazepine receptor (PBR) has been renamed the translocator protein (TSPO). In eukaryotes, the TSPO is primarily located in the outer mitochondrial membrane. In prokaryotes, it is found in the cell membrane. A broad spectrum of functions has been attributed to the TSPO, including various host defense responses, developmental processes, and mitochondrial functions. In the present review, we focus on the role of TSPO in immunological responses, apoptosis, and steroidogenesis, to determine whether these functions may be governed by a common denominator including TSPO. At physiological concentrations (nM range), the TSPO specific ligands, PK 11195 and Ro5-4864, appear to be anti-apoptotic. Knockdown of TSPO by genetic manipulation, resulting a reduction by more than 50% in [(3)H]PK 11195 binding, was reported to show anti-apoptotic effects, suggesting a potential pro-apoptotic function of TSPO. However, a reduction of more than 70% of TSPO abundance was found to cause cell death, possibly due to impairment of other essential cell functions. The pro-apoptotic function of TSPO may involve the modulation of the channel formed by the mitochondrial voltage-dependent anion channel (VDAC) and the adenine nucleotide transporter (ANT) [i.e., the mitochondrial permeability transition pore (MPTP)]. The frequently reported pro-apoptotic effects of PK 11195 and Ro5-4864 may be due to sites with low-affinity binding for these specific TSPO ligands, and not directly related to VDAC and ANT. Also at concentrations in the nM range, PK 11195 and Ro5-4864 appear to stimulate steroidogenesis. For this function TSPO by itself appears to suffice i.e. no involvement of VDAC and ANT. TSPO appears to operate as a translocator/channel to transfer cholesterol into mitochondria where it is converted to pregnenolone, a precursor of further steroidogenesis. Apoptosis and steroids play important roles in various aspects of the host defense response. Thus, our review suggests that the involvement of TSPO and its ligands in such seemingly disparate biological functions as immunological responses, apoptosis, and steroidogenesis may have a common denominator in the multi-dimensional role of TSPO in the host-defense response to disease and injury.

Biochemical, Physiological, and Pathological Aspects of the Peripheral Benzodiazepine Receptor

The PBR is a mitochondrial protein composed of at least two subunits, an approximately 30-kDa subunit that contains the site for BZs and an approximately 18-kDa subunit that binds isoquinoline carboxamide derivatives. Porphyrins and diazepam binding inhibitor are putative endogenous ligands for these receptors, which are under neural and hormonal control. Alterations in the density of PBR seem to be a sensitive indicator of stress: up-regulation after acute stress and down-regulation induced by repeated stress. PBR-specific ligands are involved in the control of cell proliferation and differentiation, and their binding is increased in some cancer tumors. Numerous studies in various endocrine organs have revealed that PBR are located in specific regions or tissues in the organs. Furthermore, PBR densities in various organs subject to hormonal control are regulated by organotropic hormones. At least in some cases, BZ ligands do not exert a specific effect in an organ, but rather modulate the well-documented effects of that particular hormone. To the best of our knowledge, BZ ligand action in peripheral tissues is dependent on recognition of PBR, which may suggest a receptor-mediated action.

Peripheral-type benzodiazepine receptors in the regulation of proliferation of MCF-7 human breast carcinoma cell line

Peripheral-type benzodiazepine receptors (PBR) have been implicated in cell proliferation. The aim of the present study was to test the effect of the PBR ligands PK 11195 and Ro 5-4864 and the central-type benzodiazepine receptor ligand clonazepam on breast carcinoma cell proliferation, using [3H] thymidine incorporation. We then carried out a study to identify where the PBR-specific ligands Ro 5-4864 and PK 11195 act in the cell cycle, using flow cytometric analysis. We found PBR expression in the malignant breast cancer tumors, representing various levels of estrogen and/or progesterone receptors, as well as in the MCF-7 breast carcinoma cell line. PK 11195 and Ro 5-4864 inhibited cell proliferation at concentrations of 10(-5) to 10(-4) M, while clonazepam (the central-type benzodiazepine receptor-specific ligand) had no effect. In this same concentration range, PK 11195 and Ro 5-4864, in contrast to clonazepam, induced an accumulation of MCF-7 cells in both the G0-G1 and G2-M phases of the cell cycle. The present study demonstrates that PBR ligands play a role in regulating cell proliferation in the human breast carcinoma cell line MCF-7.

Increase in peripheral benzodiazepine binding sites in colonic adenocarcinoma.

Binding characteristics of peripheral benzodiazepine binding sites (PBS) in membrane homogenates of samples taken at abdominal surgery from colonic adenocarcinoma and from healthy colonic tissues in 17 patients were determined. [3H]PK 11195, an isoquinoline carboxamide derivative, which exhibits high affinity to PBS, was used as a radioligand for the binding assay. A robust increase (3.1-fold) was found in membranes from the tumor sites as compared to normal control colon, whereas the affinity of [3H]PK 11195 to PBS was similar in both tissues.

Effects of Peripheral-type Benzodiazepine Receptor Antisense Knockout on MA-10 Leydig Cell Proliferation and Steroidogenesis

The peripheral-type benzodiazepine receptor (PBR) is not only widely expressed throughout the body, but it is also genetically conserved from bacteria to humans. Many functions have been attributed to it, but its primary role remains a puzzle. In the current study, we stably transfected cultures of MA-10 Leydig cells with either control or 18-kDa PBR antisense knockout plasmids. The antisense knockout vector was driven by the human enkephalin promoter, which contains two cAMP response elements, such that cAMP treatment of transfected cells could superinduce 18-kDa PBR antisense RNA transcription and, hence, down-regulate endogenous 18-kDa PBR mRNA levels. Control and knockout MA-10 cell lines were then compared at the level of receptor binding, thymidine incorporation, and steroid biosynthesis. Eighteen-kilodalton PBR knockout reduced the maximal binding capacity of tritium-labeled PBR ligands, and the affinity of receptors to the ligands remained unaltered. Additionally, 24-h accumulation of progesterone was lower in the knockout cells. Exposure of the two cell types to 8-bromo-cAMP resulted in a robust increase in steroid production. However, a complex pattern of steroid accumulation was observed, in which further progestin metabolism was indicated. The later decline in accumulated progesterone as well as the synthesis of androstenedione were different in the two cell types. At the level of cell proliferation, reduction of 18-kDa PBR mRNA showed no effect. Thus, we conclude that the 18-kDa PBR may have a more important role in steroidogenesis than in proliferation in this Leydig cell line.

PK 11195 attenuates kainic acid-induced seizures and alterations in peripheral-type benzodiazepine receptor (PBR) protein components in the rat brain

Peripheral‐type benzodiazepine receptors (PBR) are located in glial cells in the brain and in peripheral tissues. Mitochondria form the primary location for PBR. Functional PBR appear to require at least three components: an isoquinoline binding protein, a voltage‐dependent anion channel, and an adenine nucleotide carrier. In the present study, rats received intraperitoneal kainic acid injections, which are known to cause seizures, neurodegeneration, hyperactivity, gliosis, and a fivefold increase in PBR ligand binding density in the hippocampus. In the forebrain of control rats, hippocampal voltage‐dependent anion channel and adenine nucleotide carrier abundance was relatively low, while isoquinoline binding protein abundance did not differ between hippocampus and the rest of the forebrain. One week after kainic acid injection, isoquinoline binding protein abundance was increased more than 20‐fold in the hippocampal mitochondrial fraction. No significant changes were detected regarding hippocampal voltage‐dependent anion channel and adenine nucleotide carrier abundance. Pre‐treatment with the isoquinoline PK11195, a specific PBR ligand, attenuated the occurrence of seizures, hyperactivity, and increases in isoquinoline binding protein levels in the hippocampus, which usually follow kainic acid application. These data suggest that isoquinoline binding protein may be involved in these effects of kainic acid injections.

Acute and Repeated Swim Stress Effects on Peripheral Benzodiazepine Receptors in the Rat Hippocampus, Adrenal, and Kidney ☆ ☆☆

Peripheral benzodiazepine receptor (PBR) density has been found to be sensitive to stress. We set out to compare the influences of acute and repeated swim stress on behavior and PBR density. Following acute and repeated swim stress, rats were tested in an elevated plus-maze and an open-field test for anxiety levels, and tissues were collected from the adrenal gland, kidney, and hippocampus for measurements of PBR density. The acute rather than the repeated stress led to robust alterations in PBR density. The largest reduction in hippocampal and adrenal gland PBR density was found one hour after acute stress. In the hippocampus, acute stress caused a biphasic change in PBR density: a robust reduction in PBR density one hour after the acute stress and a distinct elevation in PBR density at 24 hours, while 72 hours after stress the elevation in PBR density appeared to be reduced.

Altered platelet peripheral-type benzodiazepine receptor in posttraumatic stress disorder

Peripheral-type benzodiazephine receptors (PBR) are involved in steroidogenesis and are sensitive to stress. Reduced platelet PBR density has been demonstrated in generalized anxiety disorder (GAD), but not in obsessive-compulsive disorder (OCD). We extended this observation to another anxiety disorder, namely, posttraumatic stress disorder (PTSD). Eighteen post-Persian Gulf War PTSD patients and 17 age- and sex-matched controls were included in the study. All subjects were evaluated using the Structured Clinical Interview for DSM-III-R-Patient Version. The severity of symptoms was assessed using the DSM-III-R scale for PTSD, the Impact of Event Scale, the Beck Depression Inventory, and the State-Trait Anxiety Inventory. [3H]PK 11195 was used to label platelet PBR. All psychological parameters (except trait anxiety) were higher in PTSD patients compared to controls. Decreased platelet PBR density (−62%; p <. 001) was observed in the PTSD patients compared to controls. The reduction in PBR observed in PTSD patients was in accordance with the findings in GAD patients, but differed from those obtained in OCD patients. It is possible that the receptoral downregulation is an adaptive response aimed at preventing chronic overproduction of glucocorticoids in hyperarousal states.

Peripheral-type benzodiazepine receptors: Their implication in brain disease

Benzodiazepines are well known for their therapeutic properties, which include anxiolytic, anticonvulsant, muscle‐relaxant, and hypnotic effects. Central‐type benzodiazepine receptors (CBRs), which are considered to mediate these effects, are present exclusively in the central nervous system (CNS), where they are located on the outer cell membranes of neurons. Peripheral‐type benzodiazepine receptors (PBRs) are present in peripheral tissues and also in glia cells of the CNS. Although CBRs are located primarily on the cell membranes of neurons, PBRs are particularly abundant on the membranes of mitochondria. CBR are coupled to γ‐aminobutyric acid A (GABAA) receptors, but PBRs are not. PBRs are constituted of at least three protein subunits of different molecular weights, i.e., 18‐kDa, 32‐kDa, and 30‐kDa protein subunits. PBRs have been implicated in various functions, including steroidogenesis, mitochondrial respiration, cell growth and differentiation, and responses to stress. The presence of PBRs in glia of the CNS suggests that they may be involved in glial functions in the brain. This review discusses the involvement of glial PBRs in various neurological diseases, such as degenerative brain diseases, neurotoxic insults, brain damage, brain cancer, and anxiety disorders. It is suggested that the involvement of PBRs in steroidogenesis appears to play an important role in neuroprotection. It is also suggested that PBRs may play a role in glial uptake of excitatory amino acids and their clearance from the brain, and herewith execute neuroprotective capabilities. With this review, we hope to clarify the potential of PBRs as targets for treatment of neurological disorders and prevention of brain damage. Drug Dev. Res. 50:355–370, 2000.

Modulatory action of benzodiazepines on human term placental steroidogenesis in vitro

Peripheral benzodiazepine (BZ) binding sites (PBzS) were characterized on placental explant membranes. [3H]PK 11195, an isoquinoline carboxamide derivative, which is a ligand specific for PBzS, labeled these sites with an equilibrium dissociation constant of 2.1 nM; the maximal number of binding sites was 396 fmol/mg protein. The effect of various BZ ligands and PK 11195 on the secretion of progesterone (P4) and estradiol-17 beta (E2) from human term placental explants was studied. Exposure of placental explants to low doses (10(-8) M) of Ro 5-4864, a BZ ligand which binds with high affinity to PBzS, caused a significant (P less than 0.05) increase in the secretion of P4 and E2 into the media (2.4- and 1.4-fold, respectively). On the other hand, high doses (10(-5) M) of Ro 5-4864 caused a significant (P less than 0.05) decrease in the secretion of P4 and E2 into the media. Also, exposure of explants to diazepam (10(-7) M) and PK 11195 (10(-6) M) caused a significant increase in P4 and E2 secretion into the media. In contrast, clonazepam, a BZ ligand specific for the central-type receptors, had no effect on the secretion of either steroid. The combination of diazepam (10(-7) M) or Ro 5-4864 (10(-8) M) with PK 11195 (10(-6) M) did not enhance the stimulatory effects obtained with each agent alone. The effects exerted by Ro 5-4864, PK 11195, and diazepam may be mediated via PBzS.