Peripheral-Type Benzodiazepine/Diazepam Binding Inhibitor Receptor

0163-769X/93/1402-0222$03.00/0 Endocrine Reviews Copyright © 1993 by The Endocrine Society

Vol. 14, No. 2 Printed in U.S.A.

Peripheral-Type Benzodiazepine/Diazepam Binding Inhibitor Receptor: Biological Role in Steroidogenic Cell Function * VASSILIOS PAPADOPOULOS Department of Anatomy and Cell Biology, Georgetown University Medical Center, Washington D.C. 20007 I. II. III. IV. V.

VI.

VII. VIII.

IX. X.

Introduction Pharmacological Characterization Cellular and Subcellular Distribution Structural and Molecular Characterization Endogenous (Natural) Ligands for PBR A. Diazepam binding inhibitor (DBI) B. Porphyrins C. Benzodiazepine-like molecules D. Others Role in the Regulation of Steroid Biosynthesis A. Cellular organization and intracellular regulation of the steroidogenic pathway B. Role of PBR in the regulation of steroid biosynthesis C. Regulation by endogenous PBR ligands Role in Cellular Proliferation Other Related Functions Involving PBR A. Mitochondrial respiration B. Calcium channel activity C. Regulation of hypothalamic-pituitary-adrenal function D. Role in the GABAergic regulation of CNS Hormonal Regulation of PBR Density Summary and Future Directions

azepine binding sites is present apparently in all tissues examined including the CNS (Section HI). Because of their abundance in peripheral tissues and in order to distinguish them from the GABAA or central benzodiazepine receptors, this class of benzodiazepine binding sites was named "peripheral-type benzodiazepine binding sites." Moreover, in the last 5 yr several studies reported physiological roles for these binding sites and they were then termed "peripheraltype benzodiazepine receptors" (PBRs) (5-8). However, there are a number of names that have been assigned to this receptor: 1. "Mitochondrial benzodiazepine receptor" (9,10) because of its predominant mitochondrial localization and its involvement in different mitochondrial functions. However, this name is now inappropriate since there are reports describing the presence of the receptor in other intracellular locations and on the plasma membrane (Section III). Moreover, a number of functions that rely on PBR activation by its ligands cannot be accounted for by activation of receptors located in the mitochondria. However, the organelle name could accompany the PBR term when a specialized function is studied. 2. "Mitochondrial diazepam binding inhibitor receptor" (11) because the polypeptide diazepam binding inhibitor (DBI) has been identified as the endogenous ligand for PBR since it displaces high affinity drug ligands from the receptor. Moreover, it has been shown that DBI regulates different cell functions (Sections VI and VII) via its interaction with PBR. Although the presence of other endogenous molecules, ligands for the receptor, has also been reported (Section V), there is no evidence that any of these substances triggers any biological function upon binding to PBR. Thus, since DBI was the first endogenous biologically active ligand for the receptor, the term "DBI receptor" should be considered. 3. oj-Receptors (12), following a proposed classification for all benzodiazepine receptors, and 4. r-Receptors (13). Despite the fact that PBRs also have been found in the CNS, the most widely used term "peripheral-type" will be retained to distinquish it from the "central-type" GABAA receptor that was also found in peripheral tissues. Although it has been now shown that PBR does not bind exclusively benzodiazepines and there are different classes of drugs that bind with high affinity to it (Section II), the term "benzodi-

I. Introduction

B

ENZODIAZEPINES, a class of drugs that includes compounds such as diazepam (Valium), are frequently prescribed because of their anxiolytic, anticonvulsant, musclerelaxant, and hypnotic properties. It has been shown that the pharmacological effects of benzodiazepines are mediated via a specific receptor complex located in the central nervous system (CNS); benzodiazepines bind to a domain that allosterically regulates chloride channel gating by 7-aminobutyric acid (GABA), on GABAA receptors (1-3). In 1977, while searching for specific benzodiazepine receptors, Braestrup and Squires (4) observed the presence of high density radiolabeled diazepam binding sites in the kidney. Since then, numerous papers have reported that this class of benzodiAddress requests for reprints to: V. Papadopoulos, D.Pharm, Ph.D., Department of Anatomy and Cell Biology, Georgetown University Medical Center, 3900 Reservoir Road, NW, Washington, D.C. 20007. •Supported by National Science Foundation Grant DCB-9017752 and National Institutes of Health Grant DK-43358.

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PBR AND STEROIDOGENIC CELL FUNCTION

April, 1993

azepine" receptor will also be retained for historic purposes and will be combined with the term "DBI receptor." Thus, in this manuscript and until agreement is reached between scientists working with this receptor for a common name, the term "peripheral-type benzodiazepine/DBI receptor" (PBR) will be used. II. Pharmacological Characterization PBRs were distinguished from GABAA/benzodiazepine receptors by their distinct pharmacological profile. The structures of the different classes of drugs used to characterize PBR are given in Fig. 1. Although PBR was originally described as a second binding site for the benzodiazepine diazepam (4), which binds with relatively high affinity to both PBR and GABAA receptors, the use of a series of benzodiazepine derivatives was important in determining their distinct structural specificity (5, 14-16). The benzodiazepine Ro5-4864 (4'-chlorodiazepam) binds with high affinity to the PBR from rodent species and with low affinity STRUCTURE OF PBR LIGANDS I. BENZODIAZEPINES CH,

CH,

CH,

6 RO5-4864

DIAZEPAM

& & FLUNITRAZEPAM

CLONAZEPAM

II. ISOQUINOLINE CARBOXAMIDES

CH ,

CH, CH,

CHj

;H2-CH-CON'

jCON-CH \

CM

NO,'

PK 14105

PK 11195

P K 1 4 0 6 7 / 8 (stereoisomers)

III IMIDAZOPYRIDINES

CH,

CH,

ALPIDEM

IV 2-ARYL-3-INDOLEACETAMIDES

V PORPHYRINS ;H=CH,

N(n-Hex),

H=CH2

CH, HO 2 CCH 2 CH 2

FGIN-1-27

CH 2 CH 2 CO 2 H

PROTOPORPHYRIN IX

FIG. 1. Structure of different classes of PBR ligands described in the manuscript.

223

to GABAA receptors (5, 15, 17, 18). Conversely, clonazepam and flumazenil, which bind with high affinity to GABAA receptors, exhibit extremely low affinity for PBR. One of the benzodiazepines used to characterize PBR is flunitrazepam. Flunitrazepam was initially used as a low nanomolar photoaffinity probe for GABAA receptors (19). However, despite its high nanomolar affinity for PBR, this compound was also used to photolabel PBR (20, 21). The significance of the results obtained using flunitrazepam as a probe for PBR and as an inhibitor of hormone-stimulated steroid synthesis will be discussed later (Section VLB). In 1983, Le Fur and co-workers (22-24) developed a series of isoquinoline carboxamides, which are structurally different from benzodiazepines and have much greater selectivity for PBR than for GABAA receptors. This discovery was one of the major breakthroughs in the pharmacological and molecular characterization of PBR. Today, the isoquinoline carboxamide derivative PK 11195 is perhaps the most widely used PBR probe. Quinoline propanamides, similar in structure to isoquinoline carboxamides, and which also bind with low nanomolar affinity to PBR, were then developed by the same group (25). A pair of quinoline propanamide enantiomers signified as (-)PK 14067 and (+)PK 14068 differ in their binding affinities for PBR by 2 orders of magnitude demonstrating stereospecific binding of these receptors (25). These probes are today the most useful tools for the characterization of PBR-mediated cell functions. Presented in the chronological order of their discovery, imidazopyridines were the next series of compounds developed for studying PBR. However, unlike the quinolines, imidazopyridines bind to both PBRs and to GABAA receptors (6). In this series of compounds belongs alpidem, one of the highest affinity PBR ligand thus far reported, which exhibits a dissociation constant in the picomolar range. In contrast, the imidazopyridine zolpidem exhibits a dissociation constant at the high nanomolar range. Thus, these two compounds provide another pharmacological means for determining the specificity of PBR- mediated cellular phenomena. More recently, based on the chemical structure of imidazopyridines, a new series of compounds were developed, the 2-aryl-3-indoleacetamides (26). The prototype of this series, FGIN-1-27, has an affinity close to that of alpidem. However, unlike alpidem, FGIN-1-27 has no affinity for any other receptor tested so far, i.e. GABAA, GABAB, glycine, glutamate, dopamine, serotonine, opiate, cholecystokinin, adrenergic, cannabinoid, and a-receptors (26). In addition to the PBR ligands described above, there is a long list of other compounds, essentially those containing aromatic rings, that appear to bind to PBR with different affinities. This list includes porphyrins (27), dipyridamole (28), thiazide diuretics (29), pyrethroid insecticides (30), carbamazepine (31), lidocaine (32), certain steroids (33), and dihydropyridines (34). Binding of some of these compounds to PBR is believed to indicate an association of the receptor with specific biological functions. From all the compounds developed, the benzodiazepine (Ro5-4864) and isoquinoline carboxamide (PK 11195) bindings are the most studied. From the results reported, it is

PAPADOPOULOS

224

evident that the binding domains for these two classes of compounds are not identical. Physicochemical studies on PK 11195 and Ro5-4864 binding indicated that thermodynamically PK 11195 binding is an entropy-driven process whereas that of Ro5-4864 is enthalpy-driven (24). Based on analogies with jS-adrenergic receptors, this finding suggests Ro5-4864 is an agonist whereas PK 11195 is an antagonist (24). However, there are examples where these compounds have either similar or opposite actions, an observation that contradicts this supposition and makes it impossible to label either of these two compounds as PBR agonists or antagonists. There are now a number of studies suggesting that the benzodiazepine Ro5-4864 and the isoquinoline carboxamide PK 11195 bind to different conformational states of the receptor or that the benzodiazepine binding site partially overlaps or is allosterically coupled to the isoquinoline carboxamide binding domain: 1) Chemical modification of PBR, using the histidine-modifying reagent diethylpyrocarbonate, resulted in a specific inhibition of PK 11195 binding with no effect on Ro5-4864 binding in rat kidney membranes (35, 36). In these experiments, Ro5-4864 was able to block the inactivation by diethylpyrocarbonate. 2) Unsaturated fatty acids were found to decrease the affinity of PBR for benzodiazepines without affecting PK 11195 binding (36, 37) in rat kidney membranes; and 3) protoporphyrin IX was unable to displace [3H]Ro5-4864 at micromolar concentrations, whereas this compound could displace [3H]PK 11195 binding with an inhibitory constant of 14 nM in rat vas deferens (38), thus showing differential sensitivity by the recognition sites of both classes of compounds. Species specificity of benzodiazepine and isoquinoline carboxamide binding has also been reported. A low nanomolar affinity for PK 11195 appears conserved among all species, whereas the affinities for Ro5-4864, diazepam, and flunitrazepam are highly variable (39-41). Rodent PBR exhibit the highest affinity for benzodiazepines, whereas the affinity of feline and bovine PBR for these compounds is 100 to 200 times lower than that of PK 11195 (42, 43). Initial benzodiazepine binding studies in mammalian and nonmammalian vertebrates suggested that only higher vertebrates possessed PBRs (44, 45). However, binding studies performed with isoquinoline carboxamides also identified PBRs in lower vertebrate classes, i.e. fish (46). These studies also support the suggestion that two distinct binding domains exist, one for benzodiazepines and one for isoquinoline carboxamides, the benzodiazepine domain being weakly conserved among species. The presence of PBR in more primitive eukaryotes remains to be established. However, the recent observations that the 18,000 Mr PBR exhibits notable homology with crtK (32% amino acid identity, 66% including conservative replacements), a protein found in the photosynthetic bacterium Rhodobacter capsulatus that comprises part of the carotenoidbiosynthesis gene complex (47), suggests that PBR or PBRrelated proteins may be present not only in eukaryotes but also in prokaryotes. III. Cellular and Subcellular Distribution Radioligand binding studies revealed that PBRs are found in almost all tissues examined, although their levels of expres-

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sion exhibit a very distinct pattern. This specific pattern was critical in resolving the role of PBR in the regulation of steroid biosynthesis. The greatest levels of expression were found in steroidproducing tissues such as adrenal, testis, and ovary (7, 4850). PBRs were also abundant in tissues involved with electrolyte transport such as specific segments of the nephron in the kidney (6), salivary and sweat glands (49), and choriod plexus and ependemya of the brain (51). Aside from these tissues, PBRs are expressed in many other tissues at comparable levels, whereas tissues where PBRs are in relatively low abundance include skeletal muscle, gastrointestinal tract, and much of the brain (7, 52). In the CNS, the brain contains relatively low levels of PBRs in contrast to the spinal cord where they are quite abundant (53). It appears that brain PBRs are predominantly localized in glial cells (54-56), where they are abundant, and hence it is not surprising that it was reported recently that glial cells have steroidogenic properties (11, 56, 57). Despite the low density of PBRs in CNS, a dramatic increase in PBRs density following various insults to the brain have been observed (58-60). It should be noted, however, that PBR cannot be detected in some cell types such as the chromaffin cells of the adrenal medulla (48, 61) and certain primary cultures of neuronal origin (55). Radioligand binding studies in tissue sections followed by autoradiography indicated the PBRs were primarily associated with mitochondria (49, 62, 63). Subcellular fractionation studies performed by different groups confirmed these observations (50, 62, 63). Moreover, studies by Snyder and coworkers (64) indicated that digitonin treatment of isolated mitochondria resulted in the release of both PBR and monoamine oxidase, a marker of the outer mitochondrial membrane, but not cytochrome oxidase, a marker of the inner membrane. In contrast to these reports, another group has proposed that PBRs are located on the inner mitochondrial membranes (65). This latter study was performed with lung tissue and implied that the intramitochondrial localization of PBR may vary in different tissues. A more likely explanation may be that PBR is preferentially localized at contact sites of outer and inner mitochondrial membranes; thus, groups using mitochondrial membrane preparations of variable purity would obtain different results. Although in almost all tissues examined PBRs were found associated with the mitochondria, a more recent report indicated the presence of PBR ligand binding in nuclei, Golgi, lysosomes, and peroxisomes (66). Moreover, Weissman et al. (67), using a drug ligand for PBR that exhibited a limited potential to traverse the plasma membrane, raised the intriguing possibility that PBRs may also reside at the surface of the cells. The nonmitochondrial PBR localization was also supported by the finding of Olson et al. (68) that PBR was present in red blood cells, which lack mitochondria. However, the bulk of the studies dealing with the mitochondrial localization of PBR combined with studies demonstrating a role of PBR in different mitochondrial functions (69, 70) contributed to the existing dogma that PBR is located exclu-

April, 1993

PBR AND STEROIDOGENIC CELL FUNCTION

sively in this organelle. We used a recently developed antiPBR antiserum (71) to further examine the cellular and subcellular distribution of PBR in the adrenal gland, in the testis, and in Leydig cells in vitro, using biotin-streptavidin immunoperoxidase technique and immunofluorescence combined with confocal microscopy. The immunostaining pattern observed for both in vitro cultured Leydig cells and in vivo perfused adrenocortical cells was consistent with the mitochondrial localization of PBR (61, 72). However, a prominent cell surface distribution of PBR could also be observed by confocal microscopy. The demonstration of the presence of a subset of PBR at the plasma membrane may account for actions of PBR ligands not related to mitochondrial functions. We believe that the mitogenic effects of PBR ligands on different cell types (Section VII) are mediated via this plasma membrane fraction of PBR. However, the physiological and pharmacological roles of this cell surface PBR remain to be determined. IV. Structural and Molecular Characterization The successful detergent solubilization of the mitochondrial PBR, retaining ligand binding (73, 74), together with the development of a photoaffinity probe specific for PBR, a nitrophenyl derivative of PK 11195 known as PK 14105 (Ref. 75 and Fig.l), were the two most important factors in identifying and purifying the isoquinoline carboxamide binding site of PBR. This photoactivatable probe specifically labeled, in heart tissue, a protein of 18,000 Mr which was later confirmed by other laboratories to be present in other tissues containing PBR (8, 50, 56, 76, 77). The 18,000 Mr protein has been subsequently purified by several groups (78-80) and the corresponding complementary DNA was cloned from rat adrenal (81), human histiocytic lymphoma cell line U937 (71), bovine adrenal (82) and mouse testis (M. Gamier and V. Papadopoulos, unpublished results). The primary amino acid sequence deduced for the generated cDNA nucleotide sequence predicted a 169 amino acid protein of an approximate 18,900 Mr in all species studied, with an approximately 80% homology between species. Using the human PBR cDNA as a probe the PBR gene was located in the ql3.3 region of the long arm of human chromosome 22 (71). Despite the small size of the protein, hydropathy analysis of the amino acid sequence suggested that there are five segments with the potential to span a membrane bilayer. Additionally, there appear to be only two major regions of this protein exhibiting hydrophilic character. The hydrophobic nature of this protein most likely accounts for its membrane association (i.e. mitochondria and plasma membrane). Expression studies of the rat (81) and the bovine (82) cDNA probes in transformed human embryonal kidney cells U293 and COS-7 monkey kidney cells, respectively, demonstrated that this 18,000 Mr protein contains the binding domains for PBR ligands. Moreover, expression of the rat PBR resulted in a 2-fold increase in the number of both high affinity PK 11195 and Ro5-4864 binding sites, indicating that the determinants for both isoquinoline carboxamides and benzodiazepines were present in the rat protein. The expressed bovine PBR exhibited similar isoquinoline carbox-

225

amide binding characteristics but 40-fold lower affinity for benzodiazepines. These data are in agreement with the previous studies reporting species differences in PBR benzodiazepine binding and probably reflect species-specific differences in the primary amino acid sequences of PBR. Alternatively, the differences observed in the benzodiazepine binding may be due to the presence of a protein associated with the 18,000 Mr isoquinoline carboxamide binding site that would alter its structural conformation resulting in changes of PBR binding characteristics. This possibility was further substantiated by the observations that the cells, U293 and COS-7, used for the transfection assays, contained endogenous PBRs and thus the putative PBR-associated protein(s) that might determine the structural and functional PBR microenvironment. However, the recent expression and pharmacological characterization of human PBR in the yeast Saccharomyces cerevisiae, a host cell devoid of PBR, clearly indicated that the 18,000 Mr protein contains both the isoquinoline carboxamide and benzodiazepine domains (83). As expected, the affinity of the recombinant human PBR for Ro5-4864 was significantly lower than that for PK 11195. Thus, changes in the ligand binding characteristics of PBR expressed in different mammalian and nonmammalian vertebrate tissues appear to be due to differences in the primary amino acid sequence of PBR. Nevertheless, a role of PBRassociated proteins in the PBR ligand binding specificity cannot be excluded. Other lines of evidence suggested that PBR may be associated with other proteins. Various groups reported that digitonin-solubilized, photolabeled PBR migrated as a 170,000 to 210,000 apparent Mr protein (84, 85). In addition to the 18,000 Mr protein, Riond et al. (79) identified another protein in the Mr range of 30,000 to 35,000 that could also be photolabeled with [3H]PK14105 in human cell lines, but to a lesser extent than the 18,000 protein. This protein could also be photolabeled using [3H]flunitrazepam (79). The [3H] flunitrazepam photolabeled protein of 30,000 to 35,000 Mr size has been also found in rat kidney mitochondria (20). The [3H]AHN 086 acylation of a 35,000 Mr protein in rat pineal gland (86, 87), and the presence of a 34,000 Mr benzodiazepine binding protein (88) and a 23,000 Mr isoquinoline carboxamide binding protein (84) revealed by radiation inactivation experiments, further supported the presence of a second protein associated with PBR. More recently McEnery et al. (21) presented evidence indicating that the 18,000 Mr mitochondrial PBR was associated with two proteins of 32,000 and 30,000 Mr. These proteins were identified as the voltage-dependent anion channel (VDAC) and the adenine nucleotide carrier, respectively. VDAC is a large-conductance large-diameter ion channel with thin walls formed by a /3-sheet structure and located in the outer mitochondrial membrane, especially in the junctions between outer and inner membranes (contact sites) (89). VDAC forms a slightly anion-selective channel with complex voltage dependence and has been incorrectly referred to as "mitochondrial porin" by analogy to bacterial porins (89). McEnery et al. (21) demonstrated that [3H]flunitrazepam bound to intact kidney mitochondria could be

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recovered almost entirely in purified VDAC, which could also be radiolabeled with N,N'-dicyclohexyl carbodiimide, a specific VDAC ligand. Adenine nucleotide carrier, an inner mitochondrial membrane protein, was also identified in the isolated PBR complex by immunoblot analysis. Moreover, all three proteins (PBR, VDAC, and adenine nucleotide carrier) migrate as a single peak with an apparent Mr of 50,00070,000 on a gel filtration column consistent with the hypothesis that they are subunits of the same receptor complex (21). The authors proposed the presence of a ternary complex consisting of the 18,000 Mr protein, VDAC, and adenine nucleotide carrier that form a transport assembly which interacts with both endogenous PBR ligands and drug PBR ligands, thus mediating multiple cellular functions. According to the authors' observations, isoquinoline carboxamides will bind to the 18,000 Mr protein and benzodiazepines will bind to VDAC. However, the expression studies of cloned PBR reported above and the observations that VDAC inhibitors were unable to displace benzodiazepines from PBR (38) do not support the findings of McEnery and co-workers. In contrast, the interaction of benzodiazepine binding and the adenine nucleotide carrier in rat vas deferens mitochondria has been also reported by another laboratory (38). It is evident that further work is required to determine the relationship between PBR, VDAC, and the adenine nucleotide carrier protein. It must also be determined whether and in what manner these proteins interact in the regulation of intramitochondrial cholesterol transport (Section VLB), the most well characterized biological role of PBR. V. Endogenous (Natural) Ligands for PBR A. Diazepam binding inhibitor (DBI) DBI is a 10,000 Mr polypeptide that was initially described as an intracellular protein in a variety of species (90, 91), tissues (90), and a number of cell lines (11, 92). DBI was originally purified from rat brain by monitoring its ability to displace diazepam from the allosteric modulatory sites for GABA action on GABAA receptors (93). Subsequently, proteins with identical activity to DBI were purified from both bovine and human brain; these proteins proved to be homologous with rat DBI and were termed endozepines (94). Because there is a single gene that encodes for these proteins, it has been suggested that they should be referred to as DBI (91). The amino acid sequence of DBI from rat (90, 95, 96), murine (97), bovine (94, 98, 99), porcine (100), human (98, 101), and avian (91) species is shown in Fig. 2. Recent studies have demonstrated that DBI is able to displace benzodiazepine ligands from rat adrenal PBR and that it is highly expressed and stored in adrenal cortical and testicular Leydig cells, which are also rich in PBR (92, 101-103). It should be noted that a number of different functions have been attributed to DBI: stimulation of mitochondrial steroidogenesis (104, 105), acyl-CoA-mediated fatty acid elongation (106), inhibition of the glucagon-stimulated insulin secretion from pancreatic cells (107), stimulation of monokine production by monocytes (108), and regulation of

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cell growth (72). DBI actions mediated via PBR will be discussed latter in the manuscript (Sections VI.C and VII). However, the observations on the effects of DBI on insulin secretion by pancreatic cells or on cell proliferation presented a paradox considering that DBI was originally described as an intracellular protein; no direct evidence showing DBI secretion by cells in vitro or in vivo was presented. Moreover, analysis of the DBI amino acid sequence revealed that the characteristic sequences of secreted proteins were absent (109). Furthermore, in cultured astroglial cells and in several peripheral tissues, DBI did not appear to be associated with vesicles and was not released by depolarization (110) as it was in neurons (111). Nevertheless, DBI was present in cerebrospinal fluid (90,110), and some of its effects described above implied that DBI was indeed secreted by some cell types. Furthermore, the observation that the levels of DBI were elevated in the cerebrospinal fluid of patients suffering from severe endogenous depression (112) or hepatic encephalopathy (113) indicated a possible link between the secretion of DBI and a physiopathological phenomenon. Using a method initially developed to purify and characterize DBI from tissues and cells (92), we identified DBI in rat testis interstitial fluid by immunoblot analysis (72). This finding suggests that in vivo this peptide is secreted either by the seminiferous tubules or by the interstitial compartment of the testis. The distinct immunolocalization of the peptide in Sertoli and Leydig cells (72, 114) and the finding that mRNA coding for DBI was abundant in Leydig cells and low in Sertoli cells (115), together with the metabolic labeling studies of cultured testicular cells followed by immunoprecipitation of DBI from secreted proteins (72), provide compelling evidence that DBI is made and secreted by both Sertoli and Leydig cells. Alternatively, it is possible that DBI is not truly secreted, but merely directed and anchored within the plasma membrane, alone or as part of a larger protein, then released upon proteolytic cleavage. Indeed, recent evidence strongly suggests such a possibility. Todaro and coworkers (91,116) have shown that a protein anchored to the plasma membrane of a predicted 150,000 Mr was found to contain DBI at its amino terminus, i.e. extracellularly. Microwave fixation of rat brain permitted the extraction of at least two biologically active DBI processing products: DBI33-50 [octadecaneuropeptide (ODN) (117)] and DBI17-50 [triakontatetraneuropeptide (TTN) (118)]. ODN modulates allosterically the action of GABA on the GABAA receptor in a manner inhibited by flumazenil, while TTN binds preferentially to PBR (117-121). Although ODN and TTN have not been isolated and sequenced from adrenal or testis, HPLC analysis of adrenal and testis extracts have allowed the detection of ODN- and TTN-like immunoreactivities together with several other DBI fragments (104). B. Porphyrins Snyder and co-workers (20, 27) purified a PBR inhibitory activity from different tissues, including adrenal and kidney; this activity was identified as porphyrin. Protoporphyrin IX, mesoporphyrin IX, deuteroporphyrin IX, and hemin, all being naturally occuring porphyrins, were reported to have nano-

PBR AND STEROIDOGENIC CELL FUNCTION

April, 1993

1

RAT

10

20

.E .E •E.E... .E.E... E. .

40

50

TTN

. . .H. . .K.A ...N. . .K.A.D . .RH. . .K.S . . .K. . .R

60 RAT

30

SQADFDKAAEEVKRLKTQPTDEEMLFIYSHFKQATVGDVNTDRPGLLDLK

I MURINE BOVINE PORCINE HUMAN AVIAN

227

70

Y. Y. G.Y. LKEL.GFY.

. . .I..E...M..F. ...I..E...I ...I..E...M..FT ...I.IDC..M

80

GKAKWDSWNKLKGTSKENAMKTYVEKVEELKKKYGI

S D MURINE A. .E D.. .A. ID BOVINE A. .G D.. .A. IN PORCINE A. .E D. . .A. IN HUMAN EA..LK..1...D..NAYIS.AKTMVE. AVIAN FIG. 2. DBI amino acid sequences from different mammalian species. Dots indicate amino acid residues identical to those found in the rat. The positions, in the rat sequence, of the naturally occurring processing products of DBI, TTN, and ODN are also shown.

molar affinity for PBR, whereas catabolic porphyrin metabolites were relatively ineffective (20, 27,122). Porphyrins are formed during the biosynthesis of heme, hemoglobin, and other hemoproteins. Protoporphyrin IX, the porphyrin with the highest affinity for PBR, is synthesized in the endoplasmic reticulum and then transported into the inner mitochondrial membrane where it is coordinated with an iron atom to form hemin by the enzyme ferrochelatase (123). This raises the possibility that the mechanism of translocation of protoporphyrin IX from the outer to the inner mitochondrial membrane is mediated via PBR. However, despite these initial reports on the ability of porphyrins to displace drug ligands from PBR, binding studies in different tissues demonstrated that the affinity of porphyrins for PBR ranges from not detectable to high nanomolar concentrations (7,38) depending on the ligand used. It is evident that more experiments are necessary to clarify the interaction of porphyrins with PBR. C. Benzodiazepine-like molecules Using selective extraction protocols, HPLC purification, receptor binding displacement, and selective anti-benzodiazepine antibodies, Rothstein et al. (124) have recently identified six or seven benzodiazepine-like molecules in rat and human brain. Two of these molecules competitively displaced Ro5-4864 binding from brain mitochondrial fractions. However, complete chemical characterization of these compounds has not yet been reported. These molecules may be synthesized in vivo or they may be derived from dietary sources. The biological activity and the physiological role of these naturally occurring benzodiazepines remain to be established. D. Others High and low molecular weight inhibitors of [3H]Ro54864 binding have been fractionated from antral stomach

extracts (125). Similar extracts were later found to inhibit the binding of nitrendipine to PBR (126). Purification of the bioactive component in these preparations resulted in the isolation of a 16,000 Mr protein identified as a phospholipase A2 isoenzyme (126). This protein may directly interact with PBR or generate free fatty acids that compete for PBR binding. VI. Role in the Regulation of Steroid Biosynthesis A. Cellular organization and intracellular regulation of the steroidogenic pathway Steroidogenesis begins with the conversion of cholesterol to pregnenolone in the inner mitochondrial membrane. Pregnenolone then leaves the mitochondrion to undergo enzymatic transformation in the endoplasmic reticulum that will give raise to the final steroid products. This pathway is essentially regulated by trophic hormones such as ACTH in adrenocortical cells, and LH in testicular Ley dig and ovarian cells. The peptide hormones bind to their specific membrane receptor and activate a stimulatory GTPbinding protein which, in turn, stimulates adenylate cyclase. The stimulation of adenylate cyclase results in an increase in cAMP, which is the major second messenger of this system. With the exception of Ca2+, none of the other possible second messenger systems studied has been unequivocally shown to participate in the mechanism of action of ACTH and LH. The increased cAMP levels trigger two responses: 1) protein synthesis and 2) changes in the state of phosphorylation of specific proteins resulting in modification of their function. These newly synthesized proteins, at. accelerated rates, or posttranslationally modified under the influence of the hormones, may trigger the transport of cholesterol from sites of storage or synthesis to the inner mitochondrial membrane, where C27 side chain cleavage takes place via an enzymatic reaction. This reaction is catalyzed by the C27 side chain

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PAPADOPOULOS

cleavage cytochrome P-450 enzyme, dependent on an electron transport system comprising a ferredoxin and a flavoprotein. The processes of transport of cholesterol to the mitochondria and subsequently within the mitochondria (rate-limiting step of steroidogenesis) appear to be the points at which ACTH and LH accelerate steroid synthesis ("acute stimulation"). Pregnenolone formed as the result of this reaction proceeds to the microsomal system where it is further metabolized in different steps, some of them under hormonal control. The "trophic effect" of the peptide hormones results from long-term effects and involves the maintenance of appropriate levels of enzymes and other proteins needed for steroidogenesis. Detailed reviews on the cellular organization and regulation of steroid biosynthesis have been published (127-131). It is important to note that part of the cholesterol that is used for steroidogenesis comes from plasma (lipoproteins) and part of it is synthesized de novo in the cells (132). The observation that the lipid droplets in adrenal and Leydig cells are depleted during times of increased synthesis of steroids (133) supports the well accepted idea that the stored cholesterol is used for the synthesis of steroids; this phenomenon is particularly important in mediating the acute steroid response. An alternative source of cholesterol supply was recently identified in cultured Leydig cells. In this model, much of the stored cholesterol comes from free cellular cholesterol, mainly from the plasma membrane (134). Indeed the cholesterol content of the membrane decreases after stimulating the cells to synthesize steroid hormones and increases after supplying the cells with lipoprotein cholesterol (134, 135). Increases in the plasma membrane cholesterol concentration also resulted in increased steroid synthesis in cultured adrenal cells (136). It is possible that specialized low density cholesterol-rich vesicles may transport cholesterol within the cells. However, protein-mediated cholesterol transport to mitochondria and between the mitochondrial membranes is probably the most attractive hypothesis, trophic hormones being the regulators of these processes. Support for a protein-mediated cholesterol transport is provided by various groups who have shown that the protein synthesis inhibitor cycloheximide blocks the stimulation of steroidogenesis by these hormones. This inhibition occurs at the step of intramitochondrial transport of cholesterol to the inner membrane (137-140). It is postulated that a protein(s) with a rapid turn-over is required to mediate the translocation of cholesterol within mitochondria and that the hormones may act either to increase the level of this protein(s) or to posttranslationally activate it(them). Currently a number of proteins have been identified as potential candidates for this role: 1) A 2,200 steroidogenesis activator polypeptide [SAP (141)] present in all steroidogenic tissues, the synthesis of which is cAMP-dependent and cycloheximide-sensitive (142). SAP has been shown to increase cholesterol binding to the mitochondrial P-450scc rather than intramitochondrial cholesterol transfer. 2) A 28,000 to 30,000 Mr protein found in all steroidogenic tissues (143-146). This protein and its phosphorylated counterpart are synthesized in the cytosol and transported to mitochondria under the influence of

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cAMP or tissue-specific peptide hormones (143-148). However, there is no proof yet that this protein can mediate the transfer of cholesterol from the outer to the inner mitochondrial membrane. 3) More recently, a 8,200 Mr protein that stimulates transport of cholesterol into mitochondria has been isolated from bovine adrenals. In addition, this protein also stimulates transport from the outer to the inner membrane and promotes loading of P-450scc with substrate (cholesterol) (149, 150). This protein, which was recently shown to be des-(Gly-Ile)-DBI (105), appears to be a promising candidate for intramitochondrial transport of cholesterol.

B. Role of PBR in the regulation of steroid biosynthesis The possibility that PBR plays a role in the endocrine regulation of the adrenal and the testis was first raised by Anholt et al. (151) who showed that hypophysectomy induces a significant decrease of PBR's density in both the adrenal gland and testis. Furthermore, it has been shown that treatment with a variety of steroids modulate the number of PBRs in the rat testis (152). Administration of diazepam to men increases plasma testosterone levels (153). In rats, diazepam increases plasma corticosterone levels (154) without having any significant effect on plasma testosterone levels (155). Diazepam has been found also to directly inhibit the potassium-induced increase in aldosterone production by bovine adrenal glomerulosa cells (156). These observations, as well as the findings that PBRs are found primarily on outer mitochondrial membranes and that of all tissues in which they are found, PBRs are most abundant in steroidogenic cells (Section III), suggested a possible involvement of PBR in the regulation of steroid biosynthesis. The first direct evidence of a role for benzodiazepines in steroidogenesis has been obtained by in vitro studies on decapsulated testes and interstitial cell suspensions in which diazepam and Ro5-4864 were shown to stimulate androgen production (157,158). But these authors did not demonstrate a direct action of these drugs on Leydig cells, the androgenproducing cell of the testis. Nor did they offer data to explain how these drugs act to stimulate androgen production. The use of decapsulated testes as a model is limited since it cannot take into account the regulation of Leydig cell steroidogenesis by paracrine factors of Sertoli cell origin (159,160). At the same time, we have undertaken a detailed pharmacological/endocrinological study to determine the effects of benzodiazepines on steroid biosynthesis as well as the exact locus and the mechanism of their action (8, 161). Nine different ligands with affinities for PBR that span 4 orders of magnitude were tested for their potencies to modulate steroidogenesis in the Y-l adrenocortical and MA-10 Leydig mouse tumor cell lines. The inhibition constant (Kj) to inhibit [3H]PK 11195 binding and the EC50 for steroid biosynthesis for this series of compounds showed an excellent correlation in both cell lines. The most potent ligands stimulated steroid production by about 2- and 4-fold in the Y-l and MA-10 cells, respectively. Unlike human CG (hCG)- or cAMP-activated steroidogenesis, this stimulation was not inhibited by cycloheximide. The action of PBR ligands was not additive

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PBR AND STEROIDOGENIC CELL FUNCTION

to the stimulation by ACTH, hCG, or cAMP but was additive to that of epidermal growth factor, another regulator of MA10 Ley dig cell steroidogenesis. Moreover, PBR ligands stimulated pregnenolone biosynthesis in isolated mitochondria supplied with exogenous cholesterol in a dose-dependent manner. This effect was not observed with mitoplasts (mitochondria devoided of the outer membrane). Cytochrome P-450 side chain cleavage activity, as measured by the metabolism of 22(R)-hydroxycholesterol, was not affected by PBR ligands in intact cells. Similar results were also obtained with purified rat Leydig cells, as well as rat and bovine adrenocortical cells. These studies demonstrated that PBRs are implicated in the acute stimulation of steroidogenesis possibly by mediating the entry, distribution and/or availability of cholesterol within mitochondria. It should be noted that the stimulatory effects observed using high affinity PBR ligands were further confirmed using alpidem (162) and FGIN-1-27 (26), compounds which, thus far, have the highest affinity for PBR. Parallel studies using bovine adrenal mitochondria (105, 163), human term placental tissue (164), and ovarian granulosa cells (165) as model systems further confirmed a direct involvement of PBR in steroidogenic activation. More recently we demonstrated that PBR ligands can also stimulate rat C6-2B glial cell pregnenolone formation using both intact cells and isolated mitochondria as model systems (11, 166). When we investigated the possible step(s) of the steroid biosynthetic pathway that PBR was modulating to stimulate steroid hormone production, we found that the binding of PBR ligands to the receptor resulted in a translocation of cholesterol from the outer to inner mitochondrial membranes (69). This effect was not inhibited by cycloheximide, a well characterized inhibitor of the trophic hormone-mediated intramitochondrial cholesterol translocation. Moreover, we observed that the effects of PBR ligands on mitochondrial steroidogenesis were also independent of exogenously supplied cholesterol, indicating that PBR stimulates steroidogenesis by utilizing cholesterol already located within the mitochondrial membranes. In our search for a role of PBR in trophic hormonestimulated steroid synthesis we examined the effects of different high affinity ligands of PBR on adrenocortical and Leydig cell steroidogenesis (167). Our observations indicated that hormone-stimulated steroidogenesis is not affected by the concomitant addition of high affinity PBR ligands (69, 167). However, a 25% increase of the hCG-stimulated testosterone production by Ro5-4864 in crude interstitial cells has been recently reported (158). Two other studies described the effect of benzodiazepines on ACTH-stimulated bovine adrenal steroidogenesis, one study showing inhibitory effects of diazepam, but at concentrations up to 10~3-10~4 M (168), and the other one reporting the absence of effect of diazepam on ACTH-stimulated steroidogenesis (169). Moreover, we observed that flunitrazepam, a benzodiazepine that binds to PBR with high nanomolar affinity, inhibited (by 30-60%) trophic hormone- and cAMP-stimulated steroidogenesis in both adrenocortical and Leydig cells (167). Scatchard analysis indicated the presence of one class of binding sites for

229

radiolabeled flunitrazepam, verified as being PBR by displacement studies with a series of PBR ligands. An inhibition of pregnenolone formation was also observed in isolated mitochondria, and it was further characterized as a reduction of cholesterol transport to inner mitochondrial membranes. The inhibitory effect of flunitrazepam on hormone-stimulated steroid production was reversed upon addition of higher affinity PBR ligands (167). These results therefore imply that hormone-stimulated steroidogenesis involves, at least in part, the participation of PBR at the step of intramitochondrial cholesterol transport. The antagonistic effect of flunitrazepam on hormonestimulated steroidogenesis brings into the picture the potential role of VDAC as a PBR-associated protein; as discussed above (Section IV), flunitrazepam photolabels a 30,000 Mr protein, recently identified as VDAC (21), and shown to complex with PBR. It is noteworthy that De Pinto et al. (170) demonstrated that the pore-forming protein of the outer mitochondrial membrane (VDAC) of bovine heart contained five molecules of cholesterol per polypeptide chain. Therefore, the binding of PBR ligands to the receptor could change the structural interaction of PBR with VDAC so that it would subsequently result in the release of VDAC-associated cholesterol. This attractive hypothesis, however, remains to be verified. In addition to the in vitro studies presented above, there are some in vivo findings that support the role of PBR in the regulation of steroid biosynthesis. Keim and Sigg (171) showed that stress-induced elevations in glucocorticoid plasma levels were attenuated by benzodiazepines. More recently, Guidotti and co-workers (172) demonstrated that PK 11195 and Ro5-4864 but not clonazepam dramatically increased glucocorticoid plasma levels in hypophysectomized rats. Moreover, these authors observed that the ACTHinduced glucocorticoid production in hypophysectomized animals was completely blocked by administration of PK 11195. These studies not only verify our in vitro results but also unequivocally demonstrate the crucial role of PBR in the regulation of steroid biosynthesis. The role of PBR in the regulation of the steroidogenesis is schematically summarized in Fig. 3. C. Regulation by endogenous PBR ligands The finding that mitochondrial steroidogenesis can be regulated by the occupancy of PBR with specific benzodiazepine ligands has suggested that PBR may be the target for endogenous ligands, which mediate the action of ACTH and LH and act in a manner similar to that of benzodiazepine ligands. As we mentioned above, efforts in identifying endogenous ligand(s) for PBR led to the identification of a long list of substances including benzodiazepine-like materials (124), porphyrins (20), and the polypeptide diazepam binding inhibitor (DBI) (90). As mentioned above (Section VIA), one of the proteins identified as potential mediator in the hormone-regulated steroidogenesis was a 8,200 mol wt protein mixture isolated from bovine adrenals, which stimulates pregnenolone formation in bovine adrenocortical mitochondria (149, 150).

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PAPADOPOULOS

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Steroid Products

VOrH'Endoplasmlc

FIG. 3. Schematic representation of the cellular mechanisms underlying steroid biosynthesis. Polypeptide hormones initially interact with a cell surface receptor stimulating cAMP production. Activation of the cAMP-dependent protein kinase results in the liberation of free cholesterol from different intracellular stores. This cholesterol is then transported, under the influence of cAMP, to the mitochondrion and is incorporated into the outer mitochondrial membrane. PBR is shown as mediating the rate-limiting step in steroid biosynthesis which is transport of cholesterol from outer to inner mitochondrial membranes. Once associated with the inner mitochondrial membrane cholesterol is accessible to the cytochrome P450 side-chain cleavage enzyme (P450scc) where it is converted to pregnenolone, the first intermediate in the synthesis of all steroid hormones. The endogenous ligand for PBR, DBI, and its biologically active naturally occuring processing product, TTN, are also shown.

This protein mixture was further identified to be composed of two proteins, des-(Gly-Ile)-DBI and ubiquitin (105). Since ubiquitin does not stimulate pregnenolone formation, it was infered that des-(Gly-Ile)-DBI was the active element in the process of steroid biosynthesis in bovine adrenocortical cells (105). Our data provided direct evidence that native brain DBI modulates, at nanomolar concentrations, mitochondrial steroidogenesis in adrenocortical, Ley dig, and glial cells (11, 104). The stimulation of mitochondrial pregnenolone formation by drug ligands of PBR (i.e. PK 11195) was found to be nonadditive with that of DBI. Moreover, flunitrazepam inhibited the stimulation by DBI by about 70%, suggesting that DBI mediates its action via an interaction with PBR. An important observation was that the amount of DBI found in the cells corresponded to a level 2- to 4-fold greater than that required to obtain a maximal steroidogenic response in mitochondrial preparations (104), which further supported an important physiological role for this protein. The PBR-mediated stimulatory effect of DBI on mitochondrial pregnenolone formation reflects the PBR-activated cholesterol translocation from the outer to the inner mitochondrial membrane. It should be noted that the stimulatory effect of PBR drug ligands as well as DBI seems specific for cholesterol since no effect on corticosterone production by adrenocortical mitochondria was observed, in the presence or absence of the substrate deoxycorticosterone (V. Papadopoulos, unpublished observations). One point that needs to be mentioned is the observation that while DBI was bioactive at nanomolar concentrations,

displacement studies indicated that it had an IC50 of approximately 1-5 fiM (72, 103). This discrepancy could only be explained by the differences in the experimental conditions used to study steroid formation and binding characteristics (37 C for steroid formation and 4 C for binding studies). In a more recent study we examined the capacity of DBI to displace radiolabeled benzodiazepines under the same conditions that we used to determine its steroidogenic effect. The results clearly indicated that under these conditions, DBI exhibited nanomolar affinity for PBR, in agreement with the steroidogenic potency of the protein (11). Since the studies on mitochondrial steroid formation were performed using purified rat brain DBI, we then purified and characterized the rat testis protein (72). Four different criteria were used to identify the 10,000 Mr (pi 6.7) testicular purified protein as DBI: 1) The purified protein was recognized by an anti-DBI antiserum. 2) Displacement studies using [3H]Ro54864 indicated that the displacement curve obtained was almost identical to the one published for rat brain DBI. 3) Rat testis DBI added to MA-10 Ley dig cell mitochondria stimulated pregnenolone formation in a similar fashion to that reported for rat brain DBI. 4) Amino acid sequence analysis of a 2,000 Mr fragment generated by a combined cyanogen bromide and trypsin cleavage of the protein revealed that the first 10 amino acids identified (QATVGDVNTD) were identical to ODN [DBI-(33-50)]. These results provide unequivocal evidence that the purified protein is indeed DBI and that DBI derived from either brain or testis has identical biological activity. One intriguing finding was recently made by Brown and

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PBR AND STEROIDOGENIC CELL FUNCTION

Hall (173) who observed that purified bovine adrenal des(Gly-Ile)-DBI or bovine brain DBI was able to directly stimulate the P450scc in a reconstituted enzyme system. More specifically, these authors reported that DBI increased the rate of reduction of P450scc by NADPH and the electron carriers. Similar results were also obtained using purified bovine testis DBI (A. S. Brown and V. Papadopoulos, unpublished results). These data suggest that in vivo, DBI would enter the mitochondria to activate the P450scc located on the inner membrane. However, immunolocalization of DBI in testicular Leydig cells by electron microscopy indicated that DBI was localized around the mitochondria but failed to show any specific immunoreactivity in the inner mitochondrial membrane (114). Nevertheless, the possibilities that DBI may transiently enter the mitochondria or that it may interact with the P450scc at the contact sites between outer and inner membranes remain to be investigated. In order to determine the biologically active region of DBI, chemically synthesized peptides corresponding to different amino acid sequences of the DBI molecule, including ODN and TTN, were synthesized and tested for their biological activity on mitochondrial pregnenolone formation (104). TTN specifically stimulated pregnenolone formation by adrenocortical and glial cell mitochondria with a potency and efficacy similar to that of DBI. On the other hand, ODN was less potent and less efficacious than TTN in activating pregnenolone formation in adrenocortical mitochondria and completely inactive in glial cell mitochondria. Other peptides tested were inactive. It is possible that the stimulatory effect of TTN on mitochondrial steroid biosynthesis is due to a nonspecific interaction between the a-helical structure of the peptide (120, 174) and PBR. This possibility was excluded because both DBIi7_29 and /8-endorphin, which also possess stable a-helical structures (175), failed to significantly stimulate pregnenolone formation. In further studies, incubation of adrenocortical mitochondria with labeled DBI revealed that after a 15 min incubation period, about 20% of the DBI was proteolysed to peptide products including ODN and TTN. Relevant to this finding are the observations that adrenocortical mitochondria exhibit high levels of ATP-dependent protease activity (129). Thus, this protease may be responsible for DBI degradation, and additional studies should examine whether this enzyme is important in the regulation of steroidogenesis. Earlier reports suggested that the levels of des-(Gly-Ile)DBI were increased after ACTH administration to bovine adrenocortical cells (149, 150). They did not, however, provide either quantitative data or metabolic labeling studies, which would be required to conclude that ACTH has an effect on the rate of synthesis and turnover of des-(Gly-Ile)DBI. To be consistent with the proposal that ACTH and LH/ hCG activate steroid production via stimulation of DBI synthesis, DBI levels should also be regulated by the hormones. Furthermore, to comply with the known characteristics of this pathway, the peptide would be expected to have a very short half-life. However, this postulate is not supported by the findings of our studies in which the effect of trophic hormones on the synthesis and turnover of DBI was exam-

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ined (92). In these studies, the incorporation and turnover dynamics of [35S]methionine-labeled DBI were quantified in two hormone-responsive tumor cell lines, the ACTH-responsive Y-l adrenocortical and the LH/hCG-responsive MA-10 Leydig cell line. We showed that trophic hormones exerted no effect on DBI synthesis or turnover even though, under the same conditions, steroid synthesis was significantly stimulated. Also, we found that in the presence of cycloheximide, no significant reduction in the immunodetectable levels of DBI were observed, whereas hormone-dependent steroidogenesis was dramatically inhibited. Moreover, these studies revealed that the half-life of DBI is more than 3h. Hence, these findings suggest that the role of DBI on acute steroidogenesis is not via direct hormonal modulation of the peptide. Based on these results we can also exclude the possibility that DBI is the long sought cycloheximide-sensitive protein. It has, however, been reported that trophic hormone replacement in hypophysectomized rats increased DBI levels in the adrenal (176) and in the Leydig cells (114). Increases in DBI levels in these experiments were observed only after at least 2 h subsequent to the administration of hormones. These observations suggest that hormonal regulation of DBI levels may be involved in the long-term trophic effect of the hormones on steroid biosynthesis, but not on the short-term "acute" steroid synthesis that occurs within seconds to minutes upon addition of the peptide hormones. We cannot, however, exclude the possibility that instead of acting on DBI synthesis or turnover, the peptide hormones may act by regulating the interaction of DBI with PBR. Such an interaction could trigger changes of PBR such as phosphorylation. There is in vitro evidence that PBR is a substrate of the cAMP-dependent protein kinase, the third messenger of peptide hormone action (177). This evidence is further substantiated by the identification in the carboxy-terminal part of rat PBR of a consensus sequence for cAMP-dependent phosphorylation (178). More recent studies in our laboratory further demonstrated the vital role of DBI in trophic hormone-stimulated steroidogenesis. Using anti-sense oligodeoxynucleotides for DBI we were able to dramatically reduce DBI levels in MA-10 Leydig tumor cells. Under these conditions hormone-stimulated steroid production was attenuated (N. Boujrad and V. Papadopoulos, unpublished results). These data suggest that DBI is a mediator of the trophic hormone action in steroidogenic cells. However, since DBI is not directly regulated by peptide hormones (92) one may speculate that PBR or the interaction of DBI with PBR is the site of hormone action. The plasma membrane localization of a fraction of PBR (61, 72), as well as the observation that DBI was secreted by both Leydig and Sertoli cells in culture and was present in the testicular interstitial fluid (72), prompted us to examine whether DBI may affect Leydig cell steroidogenesis when added as an extracellular signal. Rat testis DBI was found to stimulate both basal and hormone-stimulated (at submaximal concentrations of hCG) steroidogenesis. This raised the possibility that extracellular testicular DBI may have an important physiological role on spermatogenesis and male sexual

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development (72). It appears that this effect of DBI is unique to the testis since it could not be reproduced on adrenal cortical cell steroidogenesis (72). Moreover, the observation that DBI stimulates Leydig cells only, despite the presence of cell surface PBR in both adrenocortical and Leydig cells, and the finding that we were unable to block the DBI-induced steroid formation with PBR ligands, suggest that this effect of DBI may not be mediated via PBR. The biological activity of porphyrins, other endogenous ligands for PBR, on mitochondrial steroid synthesis has been also examined. Protoporphyrin IX, the most potent compound of this series, was found to be much less efficacious and potent than PK 11195, Ro5-4864, or DBI, when tested on adrenocortical and Leydig cell mitochondrial preparations (V. Papadopoulos and K. E. Krueger, unpublished observations). The potency of protoporphyrin IX was closely correlated with its ability to displace radiolabeled benzodiazepines in the same preparations (micromolar concentrations were needed). Protoporphyrin IX was found to have no effect on C6-2B glioma cell mitochondria pregnenolone formation and was unable to displace [3H]Ro5-4864 (26). The biological activity of the endogenous benzodiazepines has not yet been examined. As described earlier (Section V.D) there are a number of potential modulators of PBR that have been described. Between them is phospholipase A2 (126, 179), an enzyme responsible for the release of arachidonate from phospholipids, which can modulate PBR binding. Furthermore, the phospholipase A2 inhibitor lipocortin I was found to increase the binding affinity for PBR ligands (180). These effects on PBR binding may be due to an interaction of unsaturated fatty acids and phospholipids with PBR (36, 37). It is possible that these findings reflect a nonspecific biochemical perturbation of the receptor-ligand interaction. However, the modulatory roles of phospholipase A2 (181,182), fatty acids (183186), and phospholipid methylation (187-189) in the regulation of steroid biosynthesis suggest a functional relation between phospholipase A2-induced release of arachidonate and/or phospholipid methylation, PBR binding, and steroidogenesis. In support of this hypothesis, Strittmatter and coworkers (190) demonstrated that benzodiazepines stimulated phospholipid methylation, with a rank-order of potency characteristic of PBR specificity, on C6 glioma cells, a cell line now shown to be steroidogenic (11). VII. Role in Cellular Proliferation Differences in PBR levels in proliferating tumorigenic cells have been reported. Autoradiography of rat brain containing glioma tumors revealed a higher density of PBRs in the tumor tissue (191). Higher levels of PBRs were also found in colonic adenocarcinoma and ovarian carcinoma (192-194), when compared to normal tissues. Moreover, higher levels of PBRs were also identified in human brain glioma or astrocytoma (195). More recently, PBR ligand binding has been used as a diagnostic test to demonstrate the presence of brain tumor in patients, using positron emission tomography (196). Based on data from drug binding studies to PBR, a role for

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this receptor in cell proliferation in vitro has also been suggested. Initial studies using high concentrations of drug ligands indicated that binding to PBR could inhibit DNA synthesis and block mitogenesis in Swiss 3T3 cells (197), mouse spleen lymphocytes (198), and AKR mouse thymoma cell line (199). Despite the positive correlation between the affinities for PBR of the drugs used and their inhibitory effect on cell growth, the results obtained were perhaps not directly related to PBR itself but rather due to a nonspecific effect of the drugs, since at that time high affinity (nanomolar) receptor ligands were unavailable. In contrast, recent studies performed with high affinity ligands at concentrations close to the dissociation constant (nanomolar) of PBR indicated that the receptor plays a stimulatory role on cell growth and DNA synthesis (200, 201). Studies on the direct effects of PK 11195 and Ro5-4864 were performed on C6 glioma and Swiss 3T3 cells (201) and demonstrated that both ligands stimulated cell proliferation. Moreover, the mitogenic effect of PRL on the Nb2 lymphoma cell line was potentiated by nanomolar concentrations of PK 11195 and Ro5-4864 but was not altered by clonazepam, a benzodiazepine with extremely low affinity for PBR (200). We also undertook similar studies and confirmed the mitogenic effect of PBR ligands at nanomolar concentrations on Swiss 3T3 cells and extended these observations to show a similar effect in the MA-10 Leydig cell line (72). Moreover, we demonstrated that the endogenous PBR ligand, DBI, also had a mitogenic effect on both MA-10 Leydig and Swiss 3T3 cells at nanomolar concentrations. Furthermore, competition and reversibility studies using drugs of a wide range of affinity for PBR clearly demonstrated that the mitogenic effect of DBI on cell proliferation was mediated via PBR. It should be mentioned, however, that when micromolar concentrations of PBR drug ligands or DBI were used, a growth inhibitory effect was also observed (72).

VIII. Other Related Functions Involving PBR A. Mitochondrial respiration One of the first mitochondrial functions examined after the identification of the mitochondria as the intracellular location of PBR was the regulation of mitochondrial respiration. Drug ligands for PBR were found to increase respiratory state IV and decrease respiratory state III rates in a manner that correlated closely with the affinity of the drugs for PBR (70, 202). Thus, activation of PBR resulted in a significant decrease in the respiratory control ratio. However, the drugs used were not found to act as uncouplers or to change the adenine nucleotide flux and the ADP/O ratio. Moreover, PK 11195 and Ro5-4864 were also found to affect oxygen consumption of mouse C-1300 neuroblastoma cells and of rat brain cortex mitochondria (202). Such changes may influence the rate of cholesterol transfer and metabolism in mitochondria, an energy- and oxygen-dependent system (203).

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PBR AND STEROIDOGENIC CELL FUNCTION

B. Calcium channel activity An interaction of PBR with the dihydropyridines, nifedipine and nitrendipine, has been described (34, 38, 204). However, studying the function of PBR in the rat heart, Doble and co-workers (84) observed that PBR and dihydropyridine receptors [voltage-dependent calcium channels (205)] are separate molecules packaged in the same membrane compartment. Moreover, isoquinoline carboxamides were found to modulate voltage-dependent, but not receptoroperated, calcium channels (206). Taken together these observations suggest that although PBR is not a calcium channel, its structure is affected by the modulation of calcium channel activity. Considering the important role of calcium in the regulation of steroid synthesis (207), such a relation between calcium channel activity and PBR may play an important physiological role in steroidogenic cell function. C. Regulation of hypothalamic-pituitary-adrenal function Because of the localization of PBR in the CNS, pituitary, adrenal cortex, and testis, the effects of drug ligands for PBR on the hypothalamic-pituitary-adrenal function have also been examined. Initial studies demonstrated that benzodiazepines altered the release of GH, ACTH, PRL, and LH from the anterior pituitary gland (208). More recently, Calogero et al. (209) presented data showing that Ro5-4864 stimulated the hypothalamic-pituitary-adrenal axis by acting mainly on the central component of this axis, increasing the release of hypothalamic corticotropin releasing hormone. In contrast to this, PK 11195 acted mainly at the pituitary level. D. Role in the GABAergic regulation of CNS The pharmacological significance of PBR in steroidogenic tissues is important, considering the role of these receptors in cholesterol transport. However, with the major pharmacological action of the benzodiazepines being in the CNS we must consider the possibility that some benzodiazepines may affect behavior through binding to PBR in the CNS. How could such actions be explained? As we pointed out earlier in this review, brain PBRs are primarily found in glial cells (Sections HI and VII) and have a predominent mitochondrial localization. Recent studies demonstrated that glial cells synthesize steroids de novo (11, 56, 57, 166, 210). In fact, glial cells can convert cholesterol to 3/3-hydroxy-5-pregnene-20-one (pregnenolone) and thereby give origin to 3/3-hydroxy-5-androstene-17-one, pregnenolone sulfate, 3a-hydroxy-5a-pregnane-20-one, and 5a-pregnane-3a,21-diol-20-one which have been shown to modulate positively or negatively the GABA-gating of Cl" channels (211, 212). These steroids produced by glial cells have been termed "neurosteroids" because they are believed to be targeted exclusively to brain cells. Detailed characterization of the steroid biosynthetic apparatus in glial cells is far from complete. The cytochrome P450scc, which is the first steroid metabolizing enzyme, converting cholesterol into pregnenolone, has been identified in primary glial cell cultures and in glial cell lines and has been localized throughout much of the white matter and within cell bodies of the

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olfactory bulb, entorhinal cortex, and cingulate cortex (11, 56, 57, 166, 210). These data suggest that drugs that bind to PBR may regulate steroid production directly within the brain. The released neurosteroids then can modulate neuronal activity and brain function. This hypothesis, schematically represented in Fig. 4, was recently examined. The high affinity PBR ligand FGIN-1-27 that enters the brain was used in vivo to demonstrate that it could mimick the effects of the neurosteroid 5a-pregnane-3a,21-diol-20-one (abbreviated THDOC), a potent GABAA receptor regulator, on behavior (26). IX. Hormonal Regulation of PBR Density The localization of PBR in specific areas such as the posterior lobe of the pituitary (48), the cortical zone of the adrenal (48, 72, 73), the interstitial Leydig cells of the testis (48, 72, 73), the male vas deferens, prostate, seminal vesicles, and Cowper's glands (213), after luteinization in the granulosa cells of the ovary (214), in the epithelium and glands of the uterus (7, 215), the oviduct epithelium (7, 215), and in the tubular elements of the outer medulla of the kidney (216) indicated that expression of PBR may be under a specific

DBI (TTN)

Cholesterol

PBR

Pregnenolone | Glia ^ A r> A GABA

3aOHDHP Pregnenolone Sulfate JHDOC

Postsynaptic Neuron

cr FIG. 4. Schematic representation of PBR-mediated release of glial neurosteroids acting on the GABAA receptor function (GABA activated Cl~ channel). 3aOHDHP, 3a-hydroxy-5a-pregnane-20-one; pregnenolone sulfate, 3j8-hydroxy-5-pregnene-20-one sulfate; THDOC, 5a-pregnane-3a,21-diol-20-one.

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hormonal regulation. Different laboratories have examined the endocrine factors that affect PBR levels. Hypophysectomy caused a dramatic decrease of PBR density in the adrenal gland and testis, which was reversed upon trophic hormone replacement (151, 215, 217). In comparison, estrogen (217-219) and progesterone (220) have also been shown to produce changes in PBR levels of peripheral and central tissues, but the magnitude of these effects was not as pronounced. In the kidney, PBR levels appear to be regulated by mineralocorticoids (221) and possibly other steroids (220). Moreover, there are reports linking changes in PBR density, in central and peripheral tissues, to experimentally induced stress (222-225). However, no direct evidence of glucocorticoid involvement or other stress-induced factors in this process has been provided. It could be concluded from these studies that, in spite of the regulatory role of PBR in steroidogenesis, steroids may exert some degree of control over PBR levels in a variety of target tissues. In order to verify this hypothesis in an in vitro system, we examined the effects of the gonadotropin hCG and a variety of steroids, including progestagens, androgens, estrogens, and glucocorticoids, on PBR expression in the MA10 Ley dig cell line (J. L. Gaillard and V. Papadopoulos, unpublished results). PBR levels were measured by ligand binding and northern (RNA) blot analysis. We were unable to detect any change of PBR levels after a 24 h period of hCG stimulation. Progestagens, androgens, and estrogens also did not alter PBR expression over a 72 h period. Glucocorticoids exhibited both stimulatory and inhibitory effects on PBR expression depending on the culture conditions used. Characterization of the structure of the PBR gene should provide more definite answers on the hormonal regulation of PBR expression. The rat PBR gene was recently isolated and characterized (226). It is comprised of four exons spanning approximately 10 kilobases. However, sequencing of more than 1 kilobase of the region upstream of the transcription start point did not reveal the presence of any known consensus site for sequence-specific factors (226). Thus, there are presently no data available for the response elements that regulate PBR DNA transcription. X. Summary and Future Directions PBR, this widespread binding site for benzodiazepines, was originally considered by many to be insignificant with regard to the major pharmacological actions of these compounds. Recently, it has been shown that PBR plays an important role in steroidogenesis by regulating the entry of the substrate (cholesterol) in the steroidogenic pathway (ratelimiting step). However, the molecular mechanisms activated upon the binding of either drug or endogenous ligands to PBR remain unknown. Some hypotheses have been presented which need further examination. Uncovering these mechanisms will help us understand how cholesterol is liberated from the outer mitochondrial membrane and is moved to the inner membane where the P450scc is located. Furthermore, more work is needed in order to understand the mechanisms by which activation of PBR leads to increased DNA synthesis. DBI, porphyrins, and endogenous, naturally

occuring, benzodiazepine-like molecules are the endogenous ligands for PBR. However, the exact role of these endogenous ligands in the regulation of steroid biosynthesis by extracellular signals, such as peptide hormones and growth factors, and the mechanisms by which their own biosynthesis and metabolism are regulated remain to be elucidated. Identification and molecular cloning of the 18,000 Mr PBR protein from different species have also provided significant insight into these recognition sites. The observation that this 18,000 Mr protein shows homology with the crtK product of the carotenoid-biosynthesis gene cluster in R. capsulatus reinforces the concept of a role of PBR in steroidogenesis. This data further suggest that a series of related proteins might be found throughout prokaryotic, plant, and animal species. Since the process of steroid formation is common to many higher and lower eucaryotes, the identification of conserved amino acid sequences between different species will further help to understand the structure-function relationship of this protein. Furthermore, the determination of the amino acid sequences within PBR responsible for ligand binding will help in the development of high affinity specific ligands for PBR and in the better understanding of the hormonal regulation of steroidogenesis by endogenous PBR ligands. The identification of a 30,000 Mr protein as a functional component associated to the 18,000 Mr PBR indicates that PBR may be more complex than originally thought and that there may be more proteins functionally associated with the 18,000 Mr PBR protein. Identification, characterization, and molecular cloning of these PBR-associated proteins will help provide a better understanding of the structure of this receptor complex. From a different perspective, the finding that benzodiazepine ligands for PBR activate steroid synthesis changes many of the existing concepts on the pharmacological mechanisms of actions of these anxiolytic, anticonvulsant drugs. It also provides alternative explanations for differences observed in the pharmacological profiles and tolerance to various benzodiazepines. The use of high affinity PBR ligands able to stimulate endogenous steroid synthesis in therapeutics has already been considered and is under development (227). This last point assumes an even greater dimension and importance considering the possible cross-talk between the two benzodiazepine receptors (GABAA and PBR) in the CNS, where steroids (neurosteroids) produced by glial cells upon activation of PBR would act on the neuronal GABAA receptor. Acknowledgments I would like to thank my collaborators Drs. K. E. Krueger, A. Mukhin, A. S. Brown, J. L. Gaillard, P. Guarneri, M. Gamier, N. Boujrad, B. Oke, and C. A. Suarez-Quian for their valuable interactions and discussions. I am indebted to Drs. E. C. Costa, A. Guidotti, and M. Dym for their continuous support. I am also grateful to Drs. A. S. Brown and M. Culty for critically reading and improving the manuscript, and to Ms. E. Thompson for her expert secretarial work.

References 1. Haefely W, Kulcsar A, Mohler H, Pieri L, Pole P, Schaffner R 1975 Possible involvement of GAB A in the central actions of

April, 1993

2. 3.

4. 5. 6.

7. 8. 9. 10. 11.

12.

13. 14. 15. 16. 17.

18.

19. 20.

21.

PBR AND STEROIDOGENIC CELL FUNCTION

benzodiazepine derivatives. In: Costa E, Greengard P (eds) Advances in Biochemical Psychopharmacology. Raven Press, New York, vol 14:131-151 Costa E, Guidotti A 1979 Molecular mechanism in the receptor actions of benzodiazepines. Annu Rev Pharmacol Toxicol 19:531545 Pritchett DB, Sontheimer H, Shivers BD, Ymer S, Kettenmann H, Schofield PR, Seeburg PH 1989 Importance of a novel GABAA receptor subunit for benzodiazepine pharmacology. Nature 338:582-585 Braestrup C, Squires RF 1977 Specific benzodiazepine receptors in rat brain characterized by high-affinity [3H]diazepam binding. Proc Natl Acad Sci USA 74:3805-3809 Marangos PJ, Patel J, Boulenger JP, Clark-Rosenberg R 1982 Characterization of peripheral-type benzodiazepine binding sites in brain using [3H]Ro5-4864. Mol Pharmacol 22:26-32 Benavides J, Quarteronet D, Imbault F, Malgouris C, Uzan A, Renault C, Dubroeucq MC, Gueremy C, Le Fur G 1983 Labeling of peripheral-type benzodiazepine binding sites in the rat brain by using [3H]PK 11195, an isoquinoline carboxamide derivative: kinetic studies and autoradiographic localization. J Neurochem 41:1744-1750 Verma A, Snyder SH 1989 Peripheral type benzodiazepine receptors. Annu Rev Pharmacol Toxicol 29:307-322 Papadopoulos V, Mukhin AG, Costa E, Krueger KE 1990 The peripheral-type benzodiazepine receptor is functionally linked to Leydig cell steroidogenesis. J Biol Chem 265:3772-3779 Krueger KE, Papadopoulos V 1992 Mitochondrial benzodiazepine receptors and the regulation of steroid biosynthesis. Annu Rev Pharmacol Toxicol 32:211-237 McEnery MW 1992 The mitochondrial benzodiazepine receptor: evidence for association with the voltage-dependent anion channel. J Bioenerg Biomembr 24:63-69 Papadopoulos V, Guarneri P, Krueger KE, Guidotti A, Costa E 1992 Pregnenolone biosynthesis in C6 glioma cell mitochondria: regulation by a diazepam binding inhibitor mitochondrial receptor. Proc Natl Acad Sci USA 89:5118-5122 Langer SZ, Arbilla S 1988 Imidazopyridines as a tool for characterization of benzodiazepine receptors: a proposal for a pharmacological classification as omega receptor subtypes. Pharmacol Biochem Behav 29:763-766 Evans-Shields JM, Laird II HE 1991 Mitogenicaly-induced alteration of peripheral-type (T) receptor density on the NB2-11C cell clone. Soc Neurosci 17:1555 (Abstract) Schoemaker H, Bliss M, Yamamura HI 1981 Specific high-affinity saturable binding of [3H]Ro5-4864 to benzodiazepine binding sites in the rat cerebral cortex. Eur J Pharmacol 71:173-175 Schoemaker H, Boles RG, Horst WD, Yamamura HI 1983 Specific high-affinity binding sites for [3H]Ro5-4864 in rat brain and kidney. J Pharmacol Exp Ther 225:61-69 Wang JKT, Taniguchi T, Spector S 1984 Structural requirements for the binding of benzodiazepines to their peripheral-type sites. Mol Pharmacol 25:349-351 Gee KW, Brinton RE, McEwen BS 1988 Regional distribution of a Ro5-4864 binding site that is functionally coupled to the gammaaminobutyric acid/benzodiazepine receptor complex in rat brain. J Pharmacol Exp Ther 244:379-383 Puia G, Santi MR, Vicini S, Pritchett DB, Seeburg PH, Costa E 1989 Differences in the negative allosteric modulation of gammaaminobutyric acid (GABA) receptors elicited by 4'-chlorodiazepam and by /J-carboline 3-carboxylate ester: a study with natural and reconstituted receptors. Proc Natl Acad Sci USA 86:7275-7279 Thomas JW, Tallman JF 1981 Characterization of photoaffinity labeling of benzodiazepine binding sites. J Biol Chem 256:98389842 Snyder SH, Verma A, Trifiletti RR 1987 The peripheral-type benzodiazepine receptor: a protein of mitochondrial outer membranes utilizing porphyrins as endogenous ligands. FASEB J 1:282— 288 McEnery MW, Snowman AM, Trifiletti RR, Snyder SH 1992 Isolation of the mitochondrial benzodiazepine receptor: association

22

23

24

25

26.

27. 28. 29. 30. 31 32. 33. 34. 35.

36.

37. 38. 39 40

41.

235

with the voltage-dependent anion channel and the adenine nucleotide carrier. Proc Natl Acad Sci USA 89:3170-3174 Le Fur G, Perrier ML, Vaucher N, Imbault F, Flamier A, Uzan A, Renault C, Dubroeucq MC, Gueremy C 1983 Peripheral benzodiazepine binding sites: effects of PK 11195, l-(2-chlorophenyl)-N-(l-methylpropyl)-3-isoquinolinecarboxamide. I. In vitro studies. Life Sci 32:1839-1847 Le Fur G, Guilloux F, Rufat P, Uzan A, Renault C, Dubroeucq MC, Gueremy C 1983 Peripheral benzodiazepine binding sites: effects of PK 11195, l-(2-chlorophenyl)-N-(l-methylpropyl)-3isoquinolinecarboxamide. II. In vivo studies. Life Sci 32:1849-1856 Le Fur G, Vaucher N, Perrier ML, Flamier A, Benavides J, Renault C, Dubroeucq MC, Gueremy C, Uzan A 1983 Differentiation between two different ligands for peripheral benzodiazepine binding sites, [3H]Ro5-4864 and [3H]PK 11195, by thermodynamic studies, life Sci 33:449-457 Dubroeucq MC, Benavides J, Doble A, Guilloux F, Allam D, Vaucher N, Bertrand P, Gueremy C, Renault C, Uzan A, Le Fur G 1986 Stereoselective inhibition of the binding of [3H]PK 11195 to peripheral-type benzodiazepine binding sites by a quinolinepropanamide derivative. Eur J Pharmacol 128:269-272 Romeo E, Auta ), Kozikowsky A, Ma A, Papadopoulos V, Puia G, Costa E, Guidotti A 1992 2-Aryl-3-Indoleacetamides (FGIN1): a new class of potent and specific ligands for the mitochondrial DBI receptor. J Pharmacol Exp Ther 262:971-978 Verma A, Nye JS, Snyder SH 1987 Porphyrins are endogenous ligands for the mitochondrial (peripheral-type) benzodiazepine receptor. Proc Natl Acad Sci USA 84:2256-2260 Davies LP, Huston V 1981 Peripheral benzodiazepine binding sites in heart and their interaction with dipyridamole. Eur J Pharmacol 73:209-211 Lukeman DS, Fanestil DD 1987 Interactions of diuretics with a renal benzodiazepine binding site in rat kidney. J Pharmacol Exp Ther 241:950-955 Devaud LL, Murray TF 1988 Involvement of peripheral-type benzodiazepine receptors in the proconvulsant actions of pyrethroid insecticides. J Pharmacol Exp Ther 247:14-22 Marangos P], Post RM, Patel J, Zander K, Parma A, Weiss A 1983 Specific and potent interactions of carbamazepine with brain adenosine receptors. Eur J Pharmacol 93:175-182 Clark M, Post RM 1990 lidocaine binds with high affinity to peripheral-type benzodiazepine receptors. Eur J Pharmacol 179:473-475 Deckert J, Marangos PJ 1986 Hormonal interactions with benzodiazepine binding sites in vitro. Life Sci 39:675-683 Cantor EH, Kenessey G, Semenuk G, Spector S 1984 Interaction of calcium channel blockers with non-neuronal benzodiazepine binding sites. Proc Natl Acad Sci USA 81:1549-1552 Benavides J, Begassat F, Phan T, Tur C, Uzan A, Renault C, Dubroeucq MC, Gueremy C, Le Fur G 1984 Histidine modification with diethylpyrocarbonate induces a decrease in the binding of an antagonist, PK 11195, but not of an agonist, Ro 5-4864, of the peripheral benzodiazepine receptors. Life Sci 35:1249-1256 Skowronski R, Fanestil DD, Beaumont K 1987 Modification of the peripheral-type benzodiazepine receptor by arachidonate, diethylpyrocarbonate and thiol reagents. Eur J Pharmacol 143:305314 Beaumont K, Skowronski R, Vaughn DA, Fanestil DD 1988 Interactions of lipids with peripheral-type benzodiazepine receptors. Biochem Pharmacol 37:1009-1014 Escubedo E, Camins A, Talaveron C, Camarasa J 1992 Characterization of [3H]Ro5-4864 binding sites in rat vas deferens. J Neurochem 58:39-45 Basile AS, Klein DC, Skolnick P 1986 Characterization of benzodiazepine receptors in the bovine pineal gland: evidence for the presence of an atypical binding site. Mol Brain Res 1:127-135 Awad M, Gavish M 1987 Binding of [3H]Ro5-4864 and [3H]PK 11195 to cerebral cortex and peripheral tissues of various species: species differences and heterogeneity in peripheral benzodiazepine binding sites. J Neurochem 49:1407-1414 Parola AL, Laird II HE 1991 The bovine peripheral-type benzo-

236

PAPADOPOULOS

diazepine receptor: a receptor with low affinity for benzodiazepines. Life Sci 48:757-764 42. Benavides J, Savaki HE, Malgouris C, Laplace C, Daniel M, Begassat F, Desban M, Uzan A, Dubroeucq MC, Renault C, Gueremy C, Le Fur G 1984 Autoradiographic localization of peripheral benzodiazepione binding sites in the cat brain with [3H] PK 11195. Brain Res Bull 13:69-77 43. Benavides J, Vaucher N, Daniel M, Malgouris C, Doble A, Uzan A, Gueremy C, Le Fur G 1985 Peripheral benzodiazepine binding sites in human brain. Soc Neurosci 11:278 (Abstract) 44. Nielsen M, Braestrup C, Squires R 1978 Evidence for a late evolutionary appearance of brain-specific benzodiazepine receptors: an investigation of 18 vertebrate and 5 invertebrate species. Brain Res 141:342-346 45. Bolger GT, Weissman BA, Lueddens H, Basile AS, Mantione CR, Barrett JE, Witkin JM, Paul SM, Skolnick P 1985 Late evolutionary appearance of peripheral-type binding sites for benzodiazepines. Brain Res 338:366-370 46. Eshleman AJ, Murray TF 1989 Differential binding properties of the peripheral-type benzodiazepine ligands [3H]PK 11195 and [3H] Ro5-4864 in trout and mouse brain membranes. J Neurochem 53:494-502 47. Armstrong GA, Alberti M, Leach F, Hearst JE 1989 Nucleotide sequence, organization, and nature of the protein products of the carotenoid biosynthesis gene cluster of Rhodobacter capsulatus. Mol Gen Genet 216:254-268 48. De Souza EB, Anholt RRH, Murphy KMM, Snyder SH, Kuhar MS 1985 Peripheral-type benzodiazepine receptors in endocrine organs: autoradiographic localization in rat pituitary, adrenal and testis. Endocrinology 116:567-573 49. Anholt RRH, DeSouza EB, Oster-Granite ML, Snyder SH 1985 Peripheral-type benzodiazepine receptors: autoradiograpic localization in whole-body sections of neonatal rats. J Pharmacol Exp Ther 233:517-526 50. Antkiewicz-Michaluk L, Guidotti A, Krueger KE 1988 Molecular characterization and mitochondrial density of a recognition site for peripheral-type benzodiazepine ligands. Mol Pharmacol 34:272278 51. Benavides J, Quarteronet D, Imbault F, Malgouris C, Uzan A, Renault C, Dubroeucq MC, Gueremy C, Le Fur G 1983 Labeling of peripheral-type benzodiazepine binding sites in the rat brain by using [3H]PK 11195, an isoquinoline carboxamide derivative: kinetic studies and autoradiographic localization. J Neurochem 41:1744-1750 52. Gavish M, Katz Y, Bar-Ami S, Weizman R 1992 Biochemical, physiological and pathological aspects of the peripheral benzodiazepine receptor. J Neurochem 58:1589-1601 53. Villiger JW 1985 Characterization of peripheral-type benzodiazepine recognition sites in rat spinal cord. Neuropharmacology 24:95-98 54. Gallager DW, Mallorga P, Oertel W, Henneberry R, Tallman JF 1981 pHJDiazepam binding in mammalian central nervous system: a pharmacological characterization. J Neurosci 1:218-225 55. Syapin PJ, Skolnick P 1979 Characterization of benzodiazepine binding sites in cultured cells of neural origin. J Neurochem 32:1047-1051 56. Papadopoulos V, Guarneri P, Pan B, Krueger KE, Costa E 1991 Steroid synthesis by glial cells. In: Costa E, Paul SM (eds) Neurosteroids and Brain Function. Thieme Medical Publishers, New York, vol 8:165-170 57. Robel P, Jung-Testas I, Hu ZY, Akwa Y, Sananes N, Kabbadj K, Eychenne B, Sancho MJ, Kang KI, Zucman Morfin R, Baulieu EE 1991 Neurosteroids: biosynthesis and metabolism in cultured rodent glia and neurons. In: Costa E, Paul SM (eds) Neurosteroids and Brain Function. Thieme Medical Publishers, New York, vol 8:147-154 58. Benavides J, Fage D, Carter C, Scatton B 1987 Peripheral-type benzodiazepine receptors are a sensitive indirect index of neuronal damage. Brain Res 421:167-172 59. Kish SJ, Sperk G, Hornykiewicz O 1983 Alterations in benzodiazepine and GABA receptor binding in rat brain following systemic injection of kainic acid. Neuropharmacology 22:1303-1309

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60. Schoemaker H, Smith TL, Yamamura HI 1983 Effect of chronic ethanol consumption on central and peripheral-type benzodiazepine binding sites in mouse brain. Brain Res 258:347-350 61. Oke BO, Suarez-Quian CA, Riond J, Ferrara P, Papadopoulos V 1992 Cell surface localization of the peripheral-type benzodiazepine receptor in adrenal cortex. Mol Cell Endocrinol 87:R1-R6 62. Basile AS, Skolnick P 1986 Subcellular localization of 'peripheraltype* binding sites for benzodiazepines in rat brain. J Neurochem 46:305-308 63. Hirsch JD, Beyer CF, Malkowitz L, Beer B, Blume AL 1989 Characterization of ligand binding to mitochondrial benzodiazepine receptors. Mol Pharmacol 35:164-172 64. Anholt RRH, Pedersen PL, DeSouza EB, Snyder SH 1986 The peripheral-type benzodiazepine receptor: localization to the mitochondrial outer membrane. J Biol Chem 261:576-583 65. Mukherjee S, Das SK 1989 Subcellular distribution of 'peripheraltype* binding sites for [3H]Ro5-4864 in guinea pig lung. J Biol Chem 264:16713-16718 66. O'Beirne GB, Woods MJ, Williams DC 1990 Two subcellular locations for peripheral-type benzodiazepine acceptors in rat liver. Eur J Biochem 188:131-138 67. Weissman BA, Elson HF, Kaplan D, Newman AH 1990 Presence of peripheral benzodiazepine binding sites on primary rat skeletal fibroblasts. Eur J Pharmacol 187:369-375 68. Olson JMM, Ciliax BJ, Mancini WR, Young AB 1988 Presence of peripheral-type benzodiazepine binding sites on human erythrocyte membranes. Eur J Pharmacol 152:47-53 69. Krueger KE, Papadopoulos V 1990 Peripheral-type benzodiazepine receptors mediate translocation of cholesterol from outer to inner mitochondrial membranes in adrenocortical cells. J Biol Chem 265:15015-15022 70. Hirsch JD, Beyer CF, Malkowitz L, Beer B, Blume AJ 1989 Mitochondrial benzodiazepine receptors mediate inhibition of mitochondrial respiratory control. Mol Pharmacol 35:157-163 71. Riond J, Mattei MG, Kaghad M, Dumont X, Guillemot JC, Le Fur G, Caput D, Ferrara P 1991 Molecular cloning and chromosomal localization of a human peripheral-type benzodiazepine receptor. Eur J Biochem 195:305-311 72. Gamier M, Boujrad N, Oke BO, Brown AS, Riond J, Ferrara P, Shoyab M, Suarez-Quian CA, Papadopoulos V 1993 Diazepam binding inhibitor is a paracrine/autocrine regulator of Leydig cell proliferation and steroidogenesis. Action via peripheral-type benzodiazepine receptor and independent mechanisms. Endocrinology 132:444-458 73. Benavides J, Manager J, Burgevin MC, Ferris O, Uzan A, Gueremy C, Renault C, Le Fur G 1985 Characterization of solubilized "peripheral-type* benzodiazepine binding sites from rat adrenal gland using [3H]PK 11195, an isoquinoline carboxamide derivative. Biochem Pharmacol 34:167-170 74. Gavish M, Fares F 1985 Solubilization of peripheral benzodiazepine binding sites from rat kidney. J Neurosci 5:2889-2893 75. Doble A, Ferris O, Burgevin MC, Menager J, Uzan A, Dubroeucq MC, Renault C, Gueremy C, Le Fur G 1987 Photoaffinity labeling of peripheral-type benzodiazepine binding sites. Mol Pharmacol 31:42-49 76. Cox DA, Ellinor PT, Kirley TL, Matlib MA 1991 Identification of a 17-KDa protein associated with the peripheral-type benzodiazepine receptor in vascular and other smooth muscle types. J Pharmacol Exp Ther 258:702-709 77. Skowronski R, Fanestil DD, Beaumont K 1988 Photoaffinity labeling of peripheral-type benzodiazepine receptors in rat kidney mitochondria with [3H]PK 14105. Eur J Pharmacol 148:187-193 78. Antkiewicz-Michaluk L, Mukhin AG, Guidotti A, Krueger KE 1988 Purification and characterization of a protein associated with peripheral-type benzodiazepine binding sites. J Biol Chem 263:17317-17321 79. Riond J, Vita N, Le Fur G, Ferrara P 1989 Characterization of a peripheral-type benzodiazepine binding site in the mitochondria of Chinese hamster ovary cells. FEBS Lett 245:238-244 80. Moynagh PN, Bailey CJ, Boyce SJ, Williams DC 1991 Immunological studies on the rat peripheral-type benzodiazepine acceptor. Biochem J 275:419-425

April, 1993

PBR AND STEROIDOGENIC CELL FUNCTION

81. Sprengel R, Werner P, Seeburg PH, Mukhin AG, Santi MR, Grayson DR, Guidotti A, Krueger KE 1989 Molecular cloning and expression of cDNA encoding a peripheral-type benzodiazepine receptor. J Biol Chem 264:20415-20421 82. Parola At, Stump DG, Pepperl DJ, Krueger KE, Regan JW, Laird II HE 1991 Cloning and expression of a pharmacologically unique bovine peripheral-type benzodiazepine receptor isoquinoline binding protein. J Biol Chem 266:14082-14087 83. Riond ), Leplatois P, Laurent P, Le Fur G, Caput D, Loison G, Ferrara P 1991 Expression and pharmacological characterization of the human peripheral-type benzodiazepine receptor in yeast. Eur J Pharmacol 208:307-312 84. Doble A, Benavides J, Ferris O, Bertrand P, Menager J, Vaucher N, Burgevin MC, Uzan A, Gueremy C, Le Fur G 1985 Dihydropyridine and peripheral-type benzodiazepine binding sites: subcellular distribution and molecular size determination. Eur J Pharmacol 119:153-167 85. Parola AL, Putnam CW, Russell DH, Laird II HE 1989 Solubilization and characterization of the liver peripheral-type benzodiazepine receptor. J Pharmacol Exp Ther 250:1149-1155 86. Lueddens HWM, Newman AH, Rice KC, Skolnick P 1986 AHN 086: an irreversible ligand of peripheral benzodiazepine receptors. Mol Pharmacol 29:540-545 87. McCabe RT, Schoenheimer JA, Skolnick P, Hauck-Newman A, Rice K, Reig J-A, Klein DC 1989 [3H]AHN 086 acylates peripheral benzodiazepine receptors in the rat pineal gland. FEBS Lett 244:263-267 88. Paul SM, Kempner ES, Skolnick P 1981 In situ molecular weight determination of brain and peripheral benzodiazepine binding sites. Eur J Pharmacol 76:465-466 89. Levitt D 1990 Gramicidin, VDAC, porin and perform channels. Curr Opin Cell Biol 2:689-694 90. Costa E, Guidotti A 1991 Diazepam binding inhibitor (DBI): a peptide with multiple biological actions, life Sci 49:325-344 91. Todaro GJ, Ro9e TM, Shoyab M 1991 Human DBI (endozepine): relationship to a homologous membrane associated protein. Neuropharmacology 30:1373-1380 92. Brown AS, Hall PF, Shoyab M, Papadopoulos V 1992 Endozepine/diazepam binding inhibitor in adrenocortical and Leydig cell lines: absence of hormonal regulation. Mol Cell Endocrinol 83:19 93. Guidotti A, Forchetti CM, Corda MG, Konkel D, Bennet CD, Costa E 1983 Isolation, characterization, and purification to homogeneity of an endogenous polypeptide with agonistic action on benzodiazepine receptors. Proc Natl Acad Sci USA 80:3531-3533 94. Shoyab M, Gentry LE, Marquardt H, Todaro G 1986 Isolation and characterization of a putative endogenous benzodiazepinoid (Endozepine) from bovine and human brain. J Biol Chem 261:11968-11973 95. Mocchetti I, Einstein R, Brosius J 1986 Putative diazepam binding inhibitor peptide: cDNA clones from rat. Proc Natl Acad Sci USA 83:7221-7225 96. Mocchetti I, Santi MR 1991 Diazepam binding inhibitor: cloning and gene expression. Neuropharmacology 30:1365-1371 97. Owens GP, Sinha AK, Sikela JM, Hahn WE 1989 Sequence and expression of the murine diazepam binding inhibitor. Mol Brain Res 6:101-108 98. Marquardt H, Todaro GJ, Shoyab M 1986 Complete amino acid sequences of bovine and human endozepines. J Biol Chem 261:9727-9731 99. Mikkelsen J, Hojrup P, Nielsen PF, Roepstorff P, Knudsen J 1987 Amino acid sequence of acyl-CoA-binding protein from cow liver. Biochem J 245:857-861 100. Chen Z, Agerbeth B, Gell K, Andersson M, Mutt V, Ostenson C-G, Efendic S, Barros-Soderling J, Persson B, Jornvall H 1988 Isolation and characterization of porcine diazepam binding inhibitor, a polypeptide not only of cerebral occurence but also common in intestinal tissues and with effects on regulation of insulin release. EurJ Biochem 174:239-245 101. Gray PW, Glaister D, Seeburg PH, Guidotti A, Costa E 1986 Cloning and expression of cDNA for human diazepam binding inhibitor: a natural ligand of an allosteric regulatory site of the

102.

103. 104.

105.

106. 107.

108.

109. 110. 111. 112.

113. 114. 115.

116. 117.

118.

119. 120.

237

gamma aminobutyric acid type A receptor. Proc Natl Acad Sci USA 83:7547-7551 Papadopoulos V, Berkovich A, Krueger KE 1991 The role of diazepam binding inhibitor and its processing products at mitochondrial benzodiazepine receptors: regulation of steroid biosynthesis. Neuropharmacology 30:1417-1423 Bovolin P, Schlichting J, Miyata J, Ferrarese C, Guidotti A, Alho H 1990 Distribution and characterization of diazepam binding inhibitor (DBI) in peripheral tissues of rat. Regul Pept 29:267-281 Papadopoulos V, Berkovich A, Krueger KE, Costa E, Guidotti A 1991 Diazepam binding inhibitor (DBI) and its processing products stimulate mitochondrial steroid biosynthesis via an interaction with mitochondrial benzodiazepine receptors. Endocrinology 129:14811488 Besman MJ, Yanagibashi K, Lee TD, Kawamura M, Hall PF, Shively JE 1989 Identification of des-(Gly-Ile)-endozepine as an effector of corticotropin-dependent adrenal steroidogenesis: stimulation of cholesterol delivery is mediated by the peripheral benzodiazepine receptor. Proc Natl Acad Sci USA 86:4897-4901 Knudsen J, Hojrup P, Hansen HO, Hansen HF, Roepstorff P 1989 Acyl-CoA-binding protein in the rat. Biochem J 262:513-519 Ostenson C-G, Ahren B, Karlsson S, Sandberg E, Efendic S 1990 Effects of porcine diazepam binding inhibitor on insulin and glucagon secretion in vitro from the rat endocrine pancreas. Regul Pept 29:143-151 Taupin V, Herbelin A, Descamps-Latscha B, Zavala F 1991 Endogenous anxiogenic peptide, ODN-diazepam binding inhibitor, and benzodiazepines enhance the production of interleukin-1 and tumor necrosis factor by human monocytes. Lymphok Cytok Res 10:7-13 Randall LL, Hardy SJS, Thorn JR 1987 Export of proteins: a biochemical review. Annu Rev Microbiol 41:507-541 Roy A 1991 Cerebrospinal fluid diazepam binding inhibitor in depressed patients and normal controls. Neuropharmacology 30:1441-1444 Ferrarese C, Vaccarino F, Alho H, Mellstrom B, Costa E, Guidotti A 1987 Subcellular localization and neuronal release of diazepam binding inhibitor. J Neurochem 48:1093-1102 Barbaccia ML, Costa E, Ferrero P, Guidotti A, Roy A, Sunderland T, Pickar D, Paul SM, Goodwin FK 1986 Diazepam binding inhibitor: a brain neuropeptide present in human spinal fluid. Studies in depression, schizophrenia and Alzheimer's disease. Arch Gen Psychiatry 43:1143-1147 Rothstein JD, McKhann G, Guarneri P, Barbaccia ML, Guidotti A, Costa E 1989 Cerebrospinal fluid content of diazepam binding inhibitor in chronichepatic encephalopathy. Ann Neurol 26:57-62 Schultz R, Pelto-Huikko M, Alho H 1992 Expression of diazepam binding inhibitor-like immunoreactivity in rat testis is dependent on pituitary hormones. Endocrinology 130:3200-3206 Rheaume E, Tonon MC, Smih F, Simard J, Desy L, Vaudry H, Pelletier G 1990 Localization of the endogenous benzodiazepine ligand octadecaneuropeptide in the rat testis. Endocrinology 127:1986-1994 Webb NR, Rose TM, Malik N, Marquardt H, Shoyab M, Todaro GJ, Lee DC 1987 Bovine and human cDNA sequences encoding a putative benzodiazepine receptor ligand. DNA 6:71-79 Ferrero P, Santi MR, Conti-Tronconi B, Costa E, Guidotti A 1986 Study of an octadecaneuropeptide derived from diazepam binding inhibitor (DBI): biological activity and presence in rat brain. Proc Natl Acad Sci USA 83:827-831 Slobodyansky E, Guidotti A, Wambebe C, Berkovich A, Costa E 1989 Isolation and characterization of a rat brain triakontatetraneuropeptide, a posttranslational product of diazepam binding inhibitor: specific action at the Ro5-4864 recognition site. J Neurochem 53:1276-1284 Costa E, Berkovich A, Guidotti A 1987 The regulation of GABAergic receptors by a novel family of endogenous neuropeptides. Life Sci 41:799-803 Berkovich A, McPhie P, Campagnone M, Guidotti A, Costa E 1990 A natural processing product of rat diazepam binding inhibitor, triakontatetraneuropeptide (diazepam binding inhibitor 17-

238

PAPADOPOULOS

50) contains an a-helix, which allows discriminations between benzodiazepine binding site subtypes. Mol Pharmacol 37:164-172 121. Guidotti A, Berkovich A, Mukhin A, Costa E 1990 Diazepam binding inhibitor: response to Knudsen and Nielsen. Biochem J 265:928-929 122. Verma A, Snyder SH 1988 Characterization of porphyrin interactions with peripheral type benzodiazepine receptors. Mol Pharmacol 34:800-805 123. Jones MS, Jones OTG 1969 The structural organization of haem synthesis in rat liver mitochondria. Biochem J 113:507-514 124. Rothstein JD, Garland W, Puia G, Guidotti A, Weber RJ, Costa E 1992 Purification and characterization of naturally occuring benzodizepine receptor ligands in rat and human brain. J Neurochem 58:2102-2115 125. Mantione CR, Weissman BA, Goldman ME, Paul SM, Skolnick P 1986 Endogenous inhibitors of 4'-[3H]chlorodiazepam (Ro54864) binding to 'peripheral* sites for benzodiazepines. FEBS Lett 176:69-74 126. Mantione CR, Goldman ME, Martin B, Bolger GT, Lueddens HWM, Paul SM, Skolnick P 1988 Purification and characterization of an endogenous protein modulator of radioligand binding to peripheral-type benzodiazepine receptors and dihydropyridine Ca -channel antagonist binding sites. Biochem Pharmacol 37:339-347 127. Simpson ER, Waterman MR 1983 Regulation by ACTH of steroid hormone biosynthesis in the adrenal cortex. Can J Biochem Cell Biol 61:692-707 128. Hall PF 1984 Cellular organization for steroidogenesis. Int Rev Cytol 86:53-95 129. Kimura T 1986 Transduction of ACTH signal from plasma membrane to mitochondria in adrenocortical steroidogenesis. Effects of peptide, phospholipid, and calcium. J Steroid Biochem 25:711-716 130. Orme-Johnson NR 1990 Distinctive properties of adrenal cortex mitochondria. Biochim Biophys Acta 1020:213-231 131. Waterman MR, Simpson ER 1989 Regulation of steroid hydroxylase gene expression is multifactorial in nature. Recent Prog Horm Res 45:533-566 132. Hall PF 1988 Testicular steroid synthesis: organization and regulation. In: Knobil E, Neil J (eds) The Physiology of Reproduction. Raven Press, New York, pp 975-998 133. Christensen AK 1975 Leydig cells In: Greep RO, Astwood EB (eds) Handbook of Physiology: Endocrinology. Williams & Wilkins Co., Baltimore, vol 5:57-94 134. Freeman DA 1987 Cyclic AMP mediated modification of cholesterol traffic in Leydig tumor cells. J Biol Chem 262:13061-13068 135. Nagy L, Freeman DA 1990 Cholesterol movement between the plasma membrane and the cholesteryl ester droplets of cultured Leydig tumour cells. Biochem J 271:809-814 136. Iida S, Papadopoulos V, Hall PF 1989 The influence of exogenous free cholesterol on steroid synthesis in cultured adrenal cells. Endocrinology 124:2619-2624 137. Simpson ER, McCarthy JL, Peterson JA 1978 Evidence that the cycloheximide-sensitive site of adrenocorticotropic hormone action is in the mitochondrion. J Biol Chem 253:3135-3139 138. Crivello CF, Jefcoate CR 1980 Intracellular movement of cholesterol in rat adrenal cells. J Biol Chem 255:8144-8151 139. Privalle CT, Crivello JF, Jefcoate CR 1983 Regulation of intramitochondrial cholesterol transfer to side-chain cleavage cytochrome P-450 in rat adrenal gland. Proc Natl Acad Sri USA 80:702-706 140. Vahouny GV, Chanderbhan R, Noland BJ, Scallen TJ 1985 Cholesterol ester hydrolase and sterol carrier proteins. Endocr Res 10:473-505 141. Pedersen RC, Brownie AC 1987 Steroidogenesis activator polypeptide isolated from a rat Leydig cell tumor. Science 236:188190 142. Mertz LM, Pedersen RC 1989 The kinetics of steroidogenesis activator polypeptide in the rat adrenal cortex. J Biol Chem 264:15274-15279 143. Krueger RJ, Orme-Johnson NR 1983 Acute adrenocorticotropic hormone stimulation of adrenal corticosteroidogenesis. J Biol Chem 258:10159-10167

Vol. 14, No. 2

144. Pon LA, Orme-Johnson NR 1986 Acute stimulation of steroidogenesis in corpus luteum and adrenal cortex by peptide hormones. J Biol Chem 261:6594-6599 145. Stocco DM, Kilgore MW 1988 Induction of mitochondrial proteins in MA-10 Leydig tumour cells with human choriogonadotropin. Biochem J 249:95-103 146. Mittre H, Aunai P, Benhaim A, Leymarie P 1990 Acute stimulation of lutropin of mitochondrial protein synthesis in small luteal cells. Eur J Biochem 187:721-726 147. Stocco DM, Sodeman TC 1991 The 30-KDa mitochondrial proteins induced by hormone stimulation in MA-10 mouse Leydig tumor cells are processed from larger precursors. J Biol Chem 266:1973119738 148. Epstein LF, Orme-Johnson NR 1991 Regulation of steroid hormone biosynthesis. Identification of precursors of a phosphoprotein targeted to the mitochondrion in stimulated rat adrenal cortex cells. J Biol Chem 266:19739-19745 149. Hall PF, Papadopoulos V, Yanagibashi K 1988 On the mechanism of action of ACTH. In: Imura H, Shizume K, Yoshida S (eds) Progress in Endocrinology. Elsevier Science Publishers, Amsterdam, pp 253-258 150. Yanagibashi K, Ohno Y, Kawamura M, Hall PF 1988 The regulation of intracellular transport of cholesterol in bovine adrenal cells: purification of a novel protein. Endocrinology 123:20752082 151. Anholt RRH, DeSouza EB, Kuhar MJ, Snyder SH 1985 Depletion of peripheral-type benzodiazepine receptors after hypophysectomy in rat adrenal gland and testis. Eur J Pharmacol 110:41-46 152. Gavish M, Okun F, Weizman A, Youdim MBH 1986 Modulation of peripheral benzodiazepine binding sites following chronic estradiol treatment. Eur J Pharmacol 127:147-151 153°. Arguelles AE, Rosner J 1975 Diazepam and plasma testosterone levels. Lancet 27:607-608 154. Marc V, Marselli PL 1969 Effect of diazepam on plasma corticosterone levels in the rat. J Pharm Pharmacol 21:784-786 155. Wilkinson M, Moger W, Grovenstine D 1980 Chronic treatment with valium (diazepam) fails to affect the reproductive system of the male rat. Life Sri 27:2285-2291 156. Shibata H, Kojima I, Oyata E 1983 Diazepam inhibits potassiuminduced aldosterone secretion in adrenal glomerulosa cells. Bicohem Biophys Res Commun 116:555-562 157. Ritta MN, Campos MB, Calandra RS 1987 Effect of GABA and benzodiazepines on testicular androgen production. Life Sci 40:791-798 158. Ritta MN, Calandra RS 1989 Testicular interstitial cells as targets for peripheral benzodiazepines. Neuroendocrinology 49:262-266 159. Skinner MK 1991 Cell-cell interactions in the testis. Endocr Rev 12:45-77 160. Papadopoulos V 1991 Identification and purification of a human Sertoli cell-secreted protein (hSCSP-80) stimulating Leydig cell steroid biosynthesis. J Clin Endocrinol Metab 72:1332-1339 161. Mukhin AG, Papadopoulos V, Costa E, Krueger KE 1989 Mitochondrial benzodiazepine receptors regulate steroid biosynthesis. Proc Natl Acad Sci USA 86:9813-9816 162. Krueger KE, Papadopoulos V, Cellular role and pharmacological implications of mitochondrial benzodiazepine binding sites recognizing diazepam binding inhibitor as a putative endogenous ligand. In: Langer SZ, Morselli PL (eds) Imidazopyridines in Anxiety Disorders: A Novel Experimental and Therapeutic Approach. Raven Press, New York, in press 163. Yanagibashi K, Ohno Y, Nakamichi N, Matsui T, Hayashida K, Takamura M, Yamada K, Tou S, Kawamura M 1989 Peripheraltype benzodiazepine receptors are involved in the regulation of cholesterol side chain cleavage in adrenocortical mitochondria. J Biochem 106:1026-1029 164. Barnea ER, Fares F, Gavish M 1989 Modulatory action of benzodiazepines on human term placental steroidogenesis in vitro. Mol Cell Endocrinol 64:155-159 165. Amsterdam A, Suh BS 1991 An indurible functional peripheral benzodiazepine receptor in mitochondria of steroidogenic granulosa cells. Endocrinology 128:503-510 166. Guarneri P, Papadopoulos V, Costa E 1992 Regulation of preg-

April, 1993

167. 168.

169.

170. 171. 172.

173. 174. 175. 176. 177.

178. 179. 180. 181. 182.

183.

184.

185. 186. 187.

PBR AND STEROIDOGENIC CELL FUNCTION

nenolone synthesis in C6 glioma cells by 4'-chlorodiazepam. Proc NatlAcadSci USA 89:5113-5117 Papadopoulos V, Nowzari FB, Krueger KE 1991 Hormone-stimulated steroidogenensis is coupled to mitochondrial benzodiazepine receptors. J Biol Chem 266:3682-3687 Holloway CD, Kenyon CJ, Dowie LJ, Corvie JE, Gray CE, Fraser R 1989 Effect of the benzodiazepines diazepam, des-N-methyldiazepam and midazolam on corticosteroid biosynthesis in bovine adrenocortical cells in vitro: location of site of action. J Steroid Biochem 33:219-225 Yanagibashi K, Ohno Y, Nakamichi N, Matsui T, Hayashida K, Takamura M, Yamada K, Tou S, Kawatnura M 1989 Diazepam potentiates the corticoidogenic response of bovine adrenal fasciculata cells to dibutyryl cyclic AMP. Jpn J Pharmacol 51:347-355 De Pinto V, Benz R, Palmieri F 1989 Interaction of non-classical detergents with the mitochondrial porin. Eur J Biochem 183:179187 Keim KL, Sigg EB 1976 Plasma corticosterone and brain catecholamines in stress: effects of psychotropic drugs. Pharmacol Biochem Behav 6:79-85 Cavallaro S, Korneyev A, Guidotti A, Costa E 1992 DBI-processing products, acting at the mitochondrial DBI receptor, mediate ACTH-induced steroidogenesis in rat adrenal. Proc Natl Acad Sci USA 89:10598-10602 Brown AS, Hall PF 1991 Stimulation by endozepine of the sidechain cleavage of cholesterol in a reconstituted enzyme system. Biochem Biophys Res Commun 180:609-614 Costa E, Berkovich A, Guidotti A 1987 The regulation of GABAergic receptors by a novel family of endogenous neuropeptides. Life Sci 41:799-803 Taylor JN, Kaiser ET 1986 The structural characterization of 0endorphin and related peptide hormones and neurotransmitters. Pharmacol Rev 38:291-319 Massotti M, Slobodyansky E, Konkel D, Costa E, Guidotti A 1991 Regulation of diazepam binding inhibitor in rat adrenal gland by adrenocorticotropin. Endocrinology 129:591-596 Krueger KE, Whalin ME, Papadopoulos V 1991 Regulation of steroid synthesis in the adrenals by mitochondrial benzodiazepine receptors. In: Costa E, Paul SM (eds) Neurosteroids and Brain Function. Thieme Medical Publishers, New York, vol 8:155-160 Kemp BE, Pearson RB 1990 Protein kinase recognition sequence motifs. Trends Biochem Sci 15:342-346 Havoundjian H, Cohen RM, Paul SM, Skolnick P 1986 Differential sensitivity of 'central* and 'peripheral* type benzodiazepine receptors to Phospholipase A2. J Neurochem 46:804-811 Mihara S-I, Sakata T, Fujimoto M 1990 Interaction of lipocortin I with peripheral-type benzodiazepine binding sites. Eur J Pharmacol 189:233-236 Sullivan MHF, Cooke BA 1985 Effects of calmodulin and lipoxygenase inhibitors on LH- and LHRH-agonist-stimulated steroidogenesis in rat Leydig cells. Biochem J 232:55-59 Cooke BA, Dirami G, Chaudry L, Choi MSK, Abayasekara DRE, Phipp L 1991 Release of arachidonic acid and the effects of corticosteroids on steroidogenesis in rat testis Leydig cells. J Steroid Biochem Mol Biol 40:465-471 Lopez-Ruiz MP, Choi MSK, Rose MP, West AP, Cooke BA 1992 Direct effect of arachidonic acid on protein kinase C and LHstimulated steroidogenesis in rat Leydig cells: evidence for tonic inhibitory control of steroidogenesis by protein kinase C. Endocrinology 130:1122-1130 Dada LA, Paz C, Mele P, Solano AR, Maciel FC, Podesta EJ 1991 The cytosol as site of phosphorylation of the cyclic AMP-dependent protein kinase in adrenal steroidogenesis. J Steroid Biochem Mol Biol 39:889-896 Meikle AW, Benson SJ, Liu X-H, Boom WD, Stringham JD 1989 Nonesterified fatty acids modulate steroidogenesis in mouse Leydig cells. Am J Physiol 257:E937-E942 Goodfriend TL, Ball DL, Elliott ME, Morrison AR, Evenson MA 1991 Fatty acids are potential endogenous regulators of aldosterone secretion. Endocrinology 128:2511-2519 Milvae RA, Alila HW, Hansel W 1983 Methylation in bovine

188.

189. 190.

191.

192. 193. 194.

195. 196.

197. 198. 199. 200.

201. 202.

203.

204.

205. 206.

207.

239

luteal cells as a regulator of luteinizing hormaone action. Biol Reprod 29:849-855 Papadopoulos V, Kamtchouing P, Drosdowsky MA, Carreau S 1987 Effects of the transmethylation inhibitor S-adenosyl-homocysteine and of the methyl donor S-adenosyl-methionine on rat Leydig cell function in vitro. J Steroid Biochem 26:93-98 Nieto A, Catt KJ 1983 Hormonal activation of phospholipid methyltransferase in the Leydig cell. Endocrinology 113:758-767 Strittmatter WJ, Hirata F, Axelrod J, Mallorga P, Tallman JF, Henneberry RC 1979 Benzodiazepine and /3-adrenergic receptor ligands independently stimulate phospholipid methylation. Nature 282:857-859 Richfield EK, Ciliax BJ, Starosta-Rubinstein SR, McKeever PE, Penney JB, Young AB 1988 Comparison of [14C]deoxyglucose metabolism and peripheral benzodiazepine receptor binding in rat C6 glioma. Neurology 38:1255-1262 Katz Y, Eitan A, Amiri Z, Gavish M 1988 Dramatic increase in peripheral benzodiazepine binding sites in human colonic carcinoma as compared to normal colon. Eur J Pharmacol 148:483-484 Katz Y, Eitan A, Gavish M 1990 Increase in peripheral benzodiazepine binding sites in colonic adenocarcinoma. Oncology 47:139-142 Katz Y, Ben-Baruch G, Kllog Y, Menczer J, Gavish M 1990 Increased density of peripheral benzodiazepine-binding sites in ovarian carcinomas as compared with benign ovarian tumours and normal ovaries. Clin Sci 78:155-158 Starosta-Rubinstein SR, Ciliax B), Penney JB, McKeever PE, Young AB 1987 Imaging of a glioma using peripheral benzodiazepine receptor ligands. Proc Natl Acad Sci USA 84:891-895 Junck L, Olson JMM, Ciliax BJ, Koeppe RA, Watkins GL, Jewett DM, McKeever PE, Wieland DM, Kilbourn MR, Starosta-Rubinstein S, Mancini WR, Kuhl DE, Greenberg HS, Young AB 1989 PET imaging of human gliomas with ligands for the peripheral benzodiazepine binding site. Ann Neurol 26:752-758 Clarke GD, Ryan PJ 1980 Tranquilizers can block mitogenesis in 3T3 cells and induce differentiation in Friend cells. Nature 287:160-161 Pawlikowski M, Lyson K, Kunert-Radek J, Stepien H 1988 Effect of benzodiazepines on the proliferation of mouse spleen lymphocytes in vitro. J Neural Transm 73:161-166 Wang JKT, Morgan JI, Spector S 1984 Benzodiazepines that bind at peripheral sites inhibit cell proliferation. Proc Natl Acad Sci USA 81:753-756 Laird II HE, Gerrish KE, Duerson KC, Putnam CW, Russell DH 1989 Peripheral benzodiazepine binding sites in Nb 2 node lymphoma cells: effects on prolactin-stimulated proliferation and ornithine decarboxylase activity. Eur J Pharmacol 171:25-35 Ikezaki K, Black KL 1990 Stimulation of cell growth and DNA synthesis by peripheral benzodiazepine. Cancer Lett 49:115-120 Larcher JP, Vayssiere JL, Le Marquer FJ, Cordeau LR, Keane PE, Bachy A, Gros F, Croizat BP 1989 Effects of peripheral benzodiazepines upon the O2 consumption of neuroblastoma cells. Eur J Pharmacol 161:197-202 McNamara BC, Jefcoate CR 1990 Heterogeneous pools of cholesterol side-chain cleavage activity in adrenal mitochondria from ACTH-treated rats: differential responses to different reducing precursors. Mol Cell Endocrinol 73:123-134 Bender AS, Hertz L 1985 Pharmacological evidence that the nonneuronal diazepam binding site in primary cultures of glial cells is associated with a calcium channel. Eur J Pharmacol 110:287-288 Rink TJ, Merritt JE 1990 Calcium signalling. Curr Opin Cell Biol 2:198-205 Mestre M, Belin C, Uzan A, Renault C, Dubroeucq MC, Gueremy C, Le Fur G 1986 Modulation of voltage-operated, but not receptor-operated, calcium channels in the rabbit aorta by PK 11195, an antagonist of peripheral-type benzodiazepine receptors. J Cardiovasc Pharmacol 8:729-734 Hall PF 1987 ACTH and corticosteroidogenesis. In: Li CH (ed) Hormonal Proteins and Peptides. Academic Press, New York, vol 13:89-125

240

PAPADOPOULOS

208. Grandison L 1983 Actions of benzodiazepines on the neuroendocrine system. Neuropharmacology 22:1505-1510 209. Calogero AE, Kamilaris TC, Bernadini R, Johnson EO, Chrousos GP, Gold PW 1990 Effects of peripheral benzodiazepine receptor ligands on hypothalamic-pituitary adrenal axis function in the rat. J Pharmacol Exp Ther 253:729-737 210. Baulieu EE, Robel P 1990 Neurosteroids: a new brain function? J Steroid Biochem Mol Biol 37:395-403 211. Purdy RH, Moore PH, Morrow AL, Paul SM 1991 The 3ahydroxy ring A-reduced metabolites of progesterone and deoxycorticosterone: natural ligands of central GABAA receptors. In: Costa E, Paul SM (eds) Neurosteroids and Brain Function. Thieme Medical Publishers, New York, vol 8:95-101 212. Majewska MD 1991 Neurosteroids: GABA-agonistic and GABAantagonistic modulators of the GABAA receptor. In: Costa E, Paul SM (eds) Neurosteroids and Brain Function. Thieme Medical Publishers, New York, vol 8:109-117 213. Katz Y, Amiri Z, Weizman A, Gavish M 1990 Identification and distribution of peripheral benzodiazepine binding sites in male genital tract. Biochem Pharmacol 40:817-820 214. Bar-Ami S, Fares F, Gavish M 1991 Altered peripheral benzodiazepine receptor density in human granulosa-lutein cells in relation to follicular maturity. Mol Cell Endocrinol 82:285-291 215. Bar-Ami S, Fares F, Gavish M 1989 Effect of hypophysectomy and hormone treatment on the induction of peripheral-type benzodiazepine binding sites in female genital axis. Horm Metab Res 21:106-107 216. Regan JW, Yamamura HI, Yamada S, Roeske WR 1981 High affinity renal [3H]flunitrazepam binding: characterization, localization, and alterations in hypertension. Life Sci 28:991-998 217. Fares F, Bar-Ami S, Brandes JM, Gavish M 1987 Gonadotropinand estrogen-induced increase of peripheral-type benzodiazepine binding sites in the hypophyseal-genital axis of rats. Eur J Pharmacol 133:97-10

Vol. 14, No. 2

218. Gavish M, Okun F, Weizman A, Youdim MBH 1986 Modulation of peripheral benzodiazepine binding sites following chronic estradiol treatment. Eur J Pharmacol 127:147-151 219. Fares F, Bar-Ami S, Brandes JM, Gavish M 1988 Changes in the density of peripheral benzodiazepine binding sites in genital organs of the female rat during the oestrous cycle. J Reprod Ferril 83:619-625 220. Gavish M, Weizman A, Youdim MBH, Okun F 1987 Regulation of central and peripheral benzodiazepine receptors in progesterone-treated rats. Brain Res 409:386-390 221. Basile AS, Paul SM, Skolnick P 1985 Adrenalectomy reduces the density of peripheral-type binding sites for benzodiazepines in the rat kidney. Eur J Pharmacol 110:149-150 222. Novas ML, Medina JH, Calvo D, De Robertis E 1987 Increase of peripheral type benzodiazepine binding sites in kidney and olfactory bulb in acutely stressed rats. Eur J Pharmacol 135:243-246 223. Gonzalez-Solveyra C, Romeo HE, Rosenstein RE, Estevez AG, Cardinali DP 1988 Benzodiazepine binding sites in rat intercapsular brown adipose tissue: effect of cold environment, denervation and endocrine ablations. Life Sci 42:393-402 224. Ferrarese C, Appollonio I, Frigo M, Perego M, Piolti R, Trabucchi M, Frattola L 1990 Decreased density of benzodiazepine receptors in lymphocytes of anxious patients: reversal after chronic diazepam treatment. Acta Psychiatr Scand 82:169-173 225. Weizman A, Tanne Z, Karp L, Martfeld Y, Tyano S, Gavish M 1987 Carbamazepine up-regulates the binding of [3H]PK 11195 to platelets of epileptic patients. Eur J Pharmacol 141:471-474 226. Casalotti SO, Pelaia G, Yakovlev AG, Csikos T, Grayson DR, Krueger KE 1992 Structure of the rat gene encoding the mitochondrial benzodiazepine receptor. Gene 121:377-382 227. Costa E, Romeo E, Auta J, Papadopoulos V, Kozikowski A, Guidotti A 1991 Is there a pharmacology of brain steroidogenesis? In: Costa E, Paul SM (eds) Neurosteroids and Brain Function. Thieme Medical Publishers, New York, 8:171-176

17th Conference of European Comparative Endocrinologists, Cordoba, Spain, September 5-10, 1994 This conference will be oganized by the Department of Cell Biology of the University of Cordoba (Spain) under the auspices of the European Society for Comparative Endocrinology. The preliminary program includes invited plenary and symposium lecturers as well as sessions of free communications and/or poster presentations on all major fields of vertebrate and invertebrate comparative endocrinology, with special emphasis on cellular, molecular, and applied aspects. For further information, please contact: Prof. Dr. F. Gracia-Navarro, ESCE Conference. Departamento de Biologia Celular, Universidad de Cordoba, Avda. San Alberto Magno s/n, 14004-CORDOBA-SPAIN. Tel: 34-57-21 85 94; Fax: 34-57-21 86 34; Email: [email protected].