I. Introduction

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Benzodiazepines (BZs)² are used clinically as muscle relaxants, anticonvulsants, anxiolytics, and sedative-hypnotics. These effects are mediated primarily via the central BZ receptors (CBRs) located in the central nervous system (CNS; Braestrup and Squires, 1977; Möhler and Okada, 1977; Tallman et al., 1980). CBRs are part of a macromolecular complex that also contains a -aminobutyric acid (GABA) receptor site and a chloride ion channel (DeLorey and Olsen, 1992). This complex has been purified, its cDNA has been cloned, and its functional receptors have been expressed in Xenopus oocytes (Schofield et al., 1987). The GABA/BZ receptor complex is a hetero-oligomer composed of five subunits: -, -, -, -, and -polypeptides. BZs bind to the -subunit and facilitate the inhibitory effect obtained by GABA (DeLorey and Olsen, 1992).

BZs also bind to other receptors, located mainly in peripheral tissues and glial cells in the brain (Fig. 1), called peripheral BZ receptors (PBRs; Verma and Snyder, 1989; Gavish et al., 1992). In rats, PBRs differ from CBRs in their drug specificity: for example, the BZ clonazepam binds to CBRs with high affinity, whereas the BZ Ro 5-4864 (4'-chlorodiazepam) as well as the non-BZ ligand PK 11195 (an isoquinoline carboxamide derivative) bind to CBRs with negligible affinity. The reverse is true with regard to PBRs (Benavides et al., 1983a,b). Imidazopyridines such as alpidem bind with high affinity to both CBRs and PBRs (Langer and Arbilla, 1988). FGIN-1 (2-aryl-3-indoleacetamide) binds with high affinity to PBRs but not to CBRs (Romeo et al., 1992). The focus of the current review is the PBR.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Relative tissue distribution of membranal [3H]PK 11195 maximal binding capacity in the adult rat. Eight adult rat tissues were compared and ranked against the adrenal gland PBR density. Average values per tissue were each calculated from at least three published values, and variability was calculated as the S.E.M. (Awad and Gavish, 1987; Gavish et al., 1987; Amiri et al., 1991; Mercer et al., 1992; Weizman et al., 1992, 1997a,b; Burgin et al., 1996; Kurumaji and Toru, 1996). Ad, adrenal; Kd, kidney; He, heart; Te, testis; Ov, Ovary; Ut, uterus; Li, liver; Br, brain.

The binding of PK 11195 and Ro 5-4864 has been studied in various species (Anholt et al., 1985b; Basile et al., 1986; Cymerman et al., 1986; Awad and Gavish, 1987, 1991; Parola et al., 1991). The binding of Ro 5-4864 has been found to differ among species (Awad and Gavish, 1987; Table 1). The order of potency of Ro 5-4864 in displacement of PK 11195 binding is rat = guinea pig > cat = dog > rabbit > calf. In calf, Ro 5-4864 is three orders of magnitude less potent than PK 11195 in binding to membranal PBRs (Basile et al., 1986; Awad and Gavish, 1987; Parola et al., 1991). In humans, it was found that PK 11195 is about two orders of magnitude more potent than Ro 5-4864 in binding to PBRs (Awad and Gavish, 1991). PBR species differences obtained in the membrane-bound state are retained in the soluble state and after purification and probably are attributable to variations in the molecular structure of PBRs rather than to differences in the membrane environment (Awad and Gavish, 1989b; Sprengel et al., 1989; Parola et al., 1991; Riond et al., 1991).

Protoporphyrin IX labels PBRs with nanomolar affinity and has been suggested to be an endogenous ligand for PBRs (Snyder et al., 1987). Subcellular studies have indicated that PBRs are mitochondrial in the rat brain (Basile and Skolnick, 1986), in the rat adrenal (Anholt et al., 1986b), and in the rat testis, lung, kidney, heart, skeletal muscle, and liver (Antkiewicz-Michaluk et al., 1988a). The use of confocal microscopy further confirmed the abundant mitochondrial localization of PBRs (Garnier et al., 1993). Other groups detected PBR binding in nuclei (O'Beirne et al., 1990; Hardwick et al., 1999), Golgi apparatus, lysosomes, peroxisomes (O'Beirne et al., 1990), and plasma membrane (Oke et al., 1992). It has also been reported that PBRs are present in mature human red blood cells, which lack mitochondria (Olson et al., 1988a). Titration of isolated rat adrenal mitochondria with digitonin demonstrated that PBRs are typically located on the outer membrane of the mitochondria (Anholt et al., 1986b). Another study showed that PBRs are also located on the inner membrane of the rat lung mitochondria (Mukherjee and Das, 1989). Hence, although PBRs are located mainly on the outer membrane of the mitochondria, other localizations are also possible.

Purification of PBRs contributed to an understanding of their function. The first step for purification requires a suitable detergent that will not destroy the binding activity. PBRs have been solubilized, using various detergents, without major impairment of binding activity (Martini et al., 1983; Benavides et al., 1985; Doble et al., 1985; Gavish and Fares, 1985; Anholt et al., 1986a; Awad and Gavish, 1989a). Antkiewicz-Michaluk et al. (1988b) purified one subunit (of 18-kDa molecular mass) of the PBR from the rat adrenal through utilization of photoaffinity labeling of this protein subunit with the isoquinoline carboxamide derivative [³H]PK 14105 and subsequent solubilization of the membranes with the detergent digitonin, followed by purification with ion-exchange chromatography and reverse-phase HPLC.

PBRs are composed of at least three subunits: the binding site for isoquinolines, with a molecular mass of 18 kDa; the voltage-dependent anion channel (VDAC), with a molecular mass of 32 kDa, which binds BZs; and the adenine nucleotide carrier, with a molecular mass of 30 kDa, which also binds BZs (McEnery et al., 1992). Although isoquinolines can bind to the 18-kDa subunit alone, PBR-specific BZs require the interaction of all three subunits for binding (Garnier et al., 1994b). This complex is located on the outer and inner mitochondrial membrane contact sites (Papadopoulos et al., 1994).

The full-length cDNA for the 18-kDa subunit has been cloned from rat (Sprengel et al., 1989), humans (Riond et al., 1991), bovine (Parola et al., 1991), and mouse (Garnier et al., 1994b). Many functions have been attributed to PBRs, including a role in cell proliferation (Wang et al., 1984; Carmel et al., 1999), steroidogenesis (Papadopoulos et al., 1990; Papadopoulos, 1993; Kelly-Hershkovitz et al., 1998), calcium flow (Cantor et al., 1984; Python et al., 1993), cellular respiration (Hirsch et al., 1989), cellular immunity (Lenfant et al., 1986), and malignancy (Starosta-Rubinstein et al., 1987; Katz et al., 1990b,c; Alenfall and Batra, 1995).

PBRs are found in many tissues. In the current review, we survey the available data on the location, molecular characteristics, functions, and clinical implications of PBRs in these various tissues.

	II. The Peripheral Benzodiazepine Receptor: Molecular Identity

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The PBRs were first described (more than 20 years ago) as BZ binding sites in non-neuronal tissue (Braestrup and Squires, 1977). Nevertheless, PBRs are found not only in peripheral tissue but also in non-neuronal brain tissue (Fig. 1; Verma and Snyder, 1989; Gavish et al., 1992). Interestingly, PBR densities are high in steroidogenic tissues, in particular in the adrenal gland (Fig. 1). PBR densities in tissues such as the kidney, heart, testis, ovary, and uterus are approximately five times as low as that in the adrenal but are still one order of magnitude higher than in other tissues such as the brain (Fig. 1).

The PBR appears to be a heteromeric complex of at least three different subunits, including an isoquinoline binding subunit (18 kDa), a VDAC (32 kDa), and an adenine nucleotide carrier (30 kDa; Snyder et al., 1990; McEnery et al., 1992; Garnier et al., 1994b). Papadopoulos et al. (1997a) reported that isoquinolines that bind specifically to PBRs interact specifically with the 18-kDa subunit, whereas PBR-specific BZ ligands bind to a site consisting of both VDAC and the 18-kDa PBR subunits (Snyder et al., 1990). In studies on the topography of PBRs in the MA-10 Leydig cell mitochondrial membrane, Papadopoulos et al. (1994, 1997a) also showed that the 18-kDa PBR protein is organized in clusters of four to six molecules associated with one VDAC molecule in such a way as to favor the formation of what they called "single pores".

The cDNA for the 850-nucleotide PBR mRNA has been cloned for a number of species, including humans (Riond et al., 1991), rat (Sprengel et al., 1989; Krueger et al., 1990), mouse (Garnier et al., 1994b), and cows (Parola et al., 1991). More recently, the genes for two of these species [humans (Lin et al., 1993) and rat (Casalotti et al., 1992)] have also been partially cloned and characterized. This approximately 13-kbp gene (Lin et al., 1993) was found as a single copy in the human genome and located on chromosome 22 in the 22q13.31 band (Riond et al., 1991; Chang et al., 1992). Figure 2 shows that this gene is composed of four exons, with the first exon and half of the fourth exon being untranslated. This gene has one transcription initiation site (Casalotti et al., 1992; Lin et al., 1993). Lin et al. (1993) also reported on an alternatively spliced PBR mRNA found in human tissue. In that case, compared with the full-size form, about 10-fold more of the smaller PBR mRNA form, in which its exon 2 sequences had been spliced out, was present in a congenital lipoid adrenal hyperplasia patient. Because this shorter mRNA is unable to translate PBR protein and because it is present in humans at greater levels than the full-length PBR mRNA species in some human conditions, it will be important to establish any novel function arising from any of its resultant translated products. It is of interest that the PBR promoter in both rat and humans does not contain a TATAA box but does contain multiple Sp1 boxes (Casalotti et al., 1992; Lin et al., 1993). This is also an indication that the product of this gene has a "housekeeping gene" function. To date, no additional transcription factor regulatory sites have been identified in this promoter.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   PBR conservation throughout evolution. A number of different species were compared with the published human gene sequence. The human gene sequence was also compared with the Escherichia coli and Archeoglobus fulgidus genomes, which were available through GenBank. A significant homology was noted between all the sequences shown above, including a bacterial (E. coli) and fungal (A. fulgidus) sequence. Percentages reported below exon boxes are with respect to the published human sequence (Riond et al., 1991).

The protein domain of exon 2 has been linked to the isoquinoline binding site and part of the PBR-specific BZ binding site (Farges et al., 1994; Papadopoulos et al., 1997a). Furthermore, a cholesterol recognition/interaction site has been characterized at the carboxyl end of the protein (Li and Papadopoulos, 1998). The 18-kDa PBR subunit is highly conserved between the four species cloned (Fig. 2; Table 1). When querying GenBank for other homologies throughout evolution, we also found that this gene is quite well conserved (59% over exons 2-4), even in bacteria (Fig. 2). Lummis et al. (1991) reported the presence of BZ binding sites in the bacterium Escherichia coli. On an initial pharmacological characterization of these bacterial receptors, they claimed that these BZ receptors are distinct from both mammalian PBRs and CBRs and appear to be more closely related to those of insects. It may be that the gene sequence we found at GenBank is related to the receptor reported by Lummis and colleagues. If this is the case, its nucleotide sequence may be more related to PBR than its pharmacology. Furthermore, the fact that BZ receptors are so well conserved throughout evolution may imply that the functions of this gene must be fundamental for the cell.

Although numerous molecular studies have characterized the PBR proteins, much still needs to be undertaken to determine their basic (cellular) functions. A detailed discussion of their putative functions follows. Here, we mention a few molecular approaches that have been attempted. Efforts to generate a PBR-negative knockout mouse did not work out in that the animals died at an early embryonic stage (Papadopoulos et al., 1997b). Unfortunately, the specific reason for the early death of these fetal mice was not resolved. Nevertheless, this result does reinforce the notion that PBRs are involved in a basic cellular function that is necessary during murine fetal development. Two studies examined the effects of knockout of the 18-kDa PBR subunit gene (Papadopoulos et al., 1997b) or of 18-kDa PBR mRNA (Kelly-Hershkovitz et al., 1998) in cultured Leydig cells. Although both studies suggested the direct involvement of PBRs in Leydig cell steroidogenesis, the latter study ruled out its involvement in Leydig cell proliferation. At the level of steroidogenesis, Papadopoulos et al. (1997b) suggested that their study indicated PBR as the rate-limiting step in cholesterol transport into the mitochondria. This was not found in the antisense knockout study, which suggested that the involvement of PBR in steroidogenesis occurs later in the pathway. The reasons for the differences between the studies may reflect the different cell lines used (MA-10 versus R2C) or the different knockout approaches applied. Specifically, although PBR gene knockout totally abrogated PBR 18-kDa expression (Papadopoulos et al., 1997b), antisense RNA knockout in cultured Leydig cells resulted in only an approximately 50% reduction in basal PBR ligand binding (Kelly-Hershkovitz et al., 1998). Hence, observations generated from the antisense knockout approach, although suggestive, are not yet conclusive. Ultimately, the resolution of these apparently conflicting data and how this relates to the fundamental function of this gene product that could result in the abrogation of early fetal mouse development remains an open question.

	III. Endogenous Ligands for Peripheral Benzodiazepine Receptors

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The PBR is designated a receptor because the binding of specific ligands to it has been implicated in several physiological functions, including steroidogenesis (Papadopoulos et al., 1990), cell growth and differentiation (Wang et al., 1984), chemotaxis (Ruff et al., 1985), and mitochondrial respiratory control (Hirsch et al., 1989). However, the quest for an endogenous ligand for this receptor is still under way. When Beaumont et al. (1983) assayed ultrafiltrates of serum and urine collected from uremic patients, as well as from normal plasma, they isolated BZ-like molecules that inhibited [³H]Ro 5-4864 binding to PBR. The affinity of these molecules for PBR was 125-fold greater than that for the CBR. The molecular mass ranged between 0.5 and 1.0 kDa, but the exact identity of the inhibitor was not specified.

Mantione et al. (1988) reported the presence of both high (molecular mass > 10 kDa) and low (molecular mass < 2 kDa) materials, isolated in crude form from rat antral stomach, that inhibited the specific binding of [³H]Ro 5-4864 to PBRs. The high-molecular mass material was further purified to yield a 16 kDa protein called anthralin. This protein also inhibited the binding of [³H]nitrendipine to the dihydropyridine Ca²⁺ channel. The protein was heat and pronase sensitive and partially sensitive to trypsin, whereas its activity was enhanced by Ca²⁺ ions in a concentration-dependent fashion. It was also found that the protein had enzymatic properties similar to the phospholipase A₂ isoenzyme. These authors proposed that anthralin may be an endogenous ligand of combined interaction, with both PBR and dihydropyridine binding sites (Mantione et al., 1988). This conclusion is consistent with pharmacological and electrophysiological observations of functional coupling between the two receptor systems (Mestre et al., 1984).

Corda et al. (1984) reported the isolation and purification to homogeneity of a 104-amino-acid-residue neuropeptide that inhibited diazepam binding to the BZ binding site. Tryptic digestion of this peptide [termed diazepam-binding inhibitor (DBI)] yielded an octadecaneuropeptide (ODN) that could compete for [³H]diazepam binding, elicit proconflict responses in rodents, and antagonize the anticonflict actions of BZs (Alho et al., 1985). The proconflict effect of ODN was inhibited by Ro 15-1788 (flumazenil; ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazole-(1,5-a)-(1,4)benzodiazepine-3-carboxylate), a CBR-specific ligand (Ferrero et al., 1986b). DBI has been detected in human brain, and DBI-like immunoreactivity has been found in the cerebrospinal fluid of human volunteers (Ferrero et al., 1986a). This immunoreactivity is increased in dementia with normal-pressure hydrocephalus but not in Alzheimer's disease, multi-infarct dementia, or dementia with Parkinson's disease (Ferrero et al., 1988). DBI has also been located in peripheral tissues rich in PBRs, such as adrenal gland, testis, and kidney (Gray et al., 1986).

Papadopoulos and Brown (1995) have shown that the PBR is the key element in the regulation of cholesterol transport from intracellular stores to the inner mitochondrial membrane and that the presence of DBI is vital for steroidogenesis and stimulated cholesterol transport (Boujrad et al., 1993). In addition, DBI directly promotes loading of cholesterol to the inner mitochondrial membrane side chain cleavage cytochrome P-450 (P-450_scc) enzyme and thus starts the metabolism of cholesterol to pregnenolone (the initial step in steroidogenesis). Increased expression of DBI has been found in brain tumors (astrocytoma, glioblastoma), with the highest levels in the most neoplastic tumors (Alho et al., 1995). A close association between DBI and PBRs in ovary further emphasizes their important role in the regulation of steroid production (Alho et al., 1994). Chemical cross-linking studies of purified metabolically radiolabeled DBI to mitochondria of R2C rat Leydig tumor cells provided direct evidence that DBI specifically binds to the 18-kDa PBR protein and plays a key role in maintaining R2C constitutive steroidogenesis (Garnier et al., 1994a). Furthermore, the close localization of PBRs and DBI at the outer mitochondrial membrane has also been demonstrated by immunoelectron microscopy (Schultz et al., 1992). It can be concluded that DBI is an important candidate for an endogenous ligand to PBRs.

Other candidates for endogenous ligands to PBRs are porphyrins. Porphyrins are known to modulate enzymatic activity of several enzymes, including tryptophan pyrrolase, guanylate cyclase, and glutathione-5-transferase, and are involved with several mitochondrial proteins (Verma and Snyder, 1988). The major physiological porphyrins, protoporphyrin IX and heme, display inhibition constant (K_i) values of 20 to 50 nM for the PBR and 1000 times less at the CBR. This receptor specificity implies a physiological role for the PBR. Iron substitution of hemin with zinc results in an 8- to 10-fold reduction in the affinity for PBRs. Additionally, tin and cobalt replacement produces derivatives that are 1000 times less affinitive than those with iron. Cytochromes, which contain porphyrins, are selectively associated with the inner mitochondrial membrane and are involved in steroidogenesis. The striking association between steroid-forming tissues such as the adrenal gland and testis, which exhibit high PBR and porphyrin levels, also suggests a physiological role for the interaction of porphyrins with PBRs (Verma et al., 1987).

	IV. Role of Peripheral Benzodiazepine Receptors in Cellular Respiration

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The PBRs are found primarily on the outer mitochondrial membrane (O'Beirne and Williams, 1988; Anholt et al., 1986b). Adrenal mitochondria possess the highest density of PBR sites, kidney mitochondria have an intermediate number, and liver has very low levels (Le Fur et al., 1983; De Souza et al., 1985). The parallels between the differential binding affinities of various PBR-specific versus CBR-specific ligands in these three tissues concerning their effect on mitochondrial respiration in the same tissues have led a few investigators to propose a role for PBRs in the regulation of the mitochondrial respiratory chain (Hirsch et al., 1989; Moreno-Sánchez et al., 1991). Hirsch et al. (1989) interpreted their data to suggest that the PBR ligands they used increased state IV and decreased state III respiration rates, which resulted in a significant decrease in the respiratory control ratio. Another group suggested that part of this inhibition of respiration was a specific effect, whereas part was nonspecific (Moreno-Sánchez et al., 1991). In particular, these authors claimed that the inhibition of reduced nicotinamide-adenine dinucleotide oxidase and the hydrolysis of ATP by PBR ligands was not specific, whereas oxidizable substrate transport was specifically inhibited by the PBR ligand AHN 086 (Moreno-Sánchez et al., 1991). In contrast, Zisterer et al. (1992) suggested that the results of those previous studies were due to nonspecific effects of PBR ligands and were not mediated through PBRs. It seems that at the present, no study definitively allows us to conclude how PBRs are actually involved in cellular respiration. If it should turn out that the PBR-specific ligands do affect mitochondrial respiration independent of PBRs, it would be essential to determine how they do this. It may be that some "alternate" peripheral-type BZ binding sites contribute to other functions or actions previously attributed to PBRs.

	V. Peripheral Benzodiazepine Receptors in Endocrine System and Steroid Regulation

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The biosynthesis of steroids in all steroidogenic tissues begins with the enzymatic conversion of the precursor cholesterol to form pregnenolone. This reaction is catalyzed by the enzyme P-450_scc, which is located on the matrix side of the inner mitochondrial membrane. The rate-limiting step in this process is the transport of cholesterol from the cellular stores across the aqueous intermembrane space of the mitochondria to the inner mitochondrial membrane and the P-450_scc. PBRs have been suggested to play a major role in this mitochondrial cholesterol transport (Krueger and Papadopoulos, 1990).

It has been reported that the steroidogenic acute regulatory protein (StAR) is involved in the acute trophic hormone regulation of steroid synthesis (Miller, 1995; Stocco and Clark, 1996a,b); however, the question as to how the transfer of cholesterol occurs remains open, and it is very likely that other proteins are involved in this process. In fact, a recent report suggests that PBR and StAR work together in cholesterol transport into the mitochondria (Sridaran et al., 1999). Two important observations suggest that PBRs are likely to play a role in steroidogenesis. First, PBRs are found primarily on the outer mitochondrial membrane (Anholt et al., 1986b; Mukherjee and Das, 1989; O'Beirne et al., 1990); second, PBRs are extremely abundant in steroidogenic endocrine tissues (Benavides et al., 1983a; De Souza et al., 1985).

It has been demonstrated that PBR-specific ligands indeed regulate mitochondrial steroidogenesis. DBI, a potential endogenous ligand for PBRs with a molecular mass of 10 kDa regulates steroidogenesis activated by corticotropin (ACTH) and luteinizing hormone via binding to PBRs and thus control mitochondrial cholesterol transport (Hall, 1991; Papadopoulos et al., 1991a,b; Brown et al., 1992). The addition of DBI to a cholesterol side chain cleavage reconstituted enzyme system was able to stimulate the conversion of cholesterol into pregnenolone (Brown and Hall, 1991), whereas the depletion of DBI from Leydig tumor cells resulted in a loss of trophic hormone-stimulated steroid production in these cells (Boujrad et al., 1993). Flunitrazepam, a partial BZ agonist for PBRs, inhibits hormone-stimulated steroid biosynthesis in Y-1 adrenocortical and MA-10 Leydig cells, via its binding to PBRs (Papadopoulos et al., 1991c). Ligands for the PBR enhance steroid production in Y-1 adrenocortical and MA-10 Leydig cell lines. These effects are attributable to their binding to PBRs (Mukhin et al., 1989; Papadopoulos et al., 1990). Ligands with high affinity to PBRs stimulate pregnenolone synthesis in brain mitochondrial preparation (Guarneri et al., 1992; Papadopoulos et al., 1992; McCauley et al., 1995). It has been suggested that PBRs play a role in the translocation of cholesterol from the outer to the inner mitochondrial membrane, which is the rate-limiting step in steroidogenesis (Krueger and Papadopoulos, 1990; Papadopoulos et al., 1990). The above findings demonstrate that PBRs play a significant role in steroid biosynthesis in various tissues.

PBRs have been characterized in the genital tract of mature female rats (Fares et al., 1987).

1. Ovary. By means of immunocytochemical, in situ hybridization, and autoradiographic methods, it has been demonstrated that the PBR and its putative endogenous ligand (DBI) are present in all ovarian steroid-secreting cells (Toranzo et al., 1994). In the immature rat ovary, PBR density increases with age (Fares et al., 1987), but hypophysectomy reduced this PBR density by 68% (Bar-Ami et al., 1989; Table 2). This effect was abolished either by 2 days' treatment with pregnant mare serum gonadotropin (PMSG) or 4 days' treatment with diethylstilbestrol (DES), a synthetic derivative of estradiol (Bar-Ami et al., 1989; Table 2). [³H]PK 11195 binding in the ovary of hypophysectomized rats was increased by 3.4- and 4-fold after PMSG or DES treatment, respectively (Bar-Ami et al., 1989; Table 2). In the intact immature rat, PMSG or DES also significantly increases PBR density (Fares et al., 1987; Table 2). PMSG, which contains both follicle-stimulating and luteinizing hormone activity (Moore and Ward, 1980), acts directly on the ovary to induce multiple follicular growth and development in the immature rat and elicits a significant increase in ovarian estradiol biosynthesis (Braw and Tsafriri, 1980).

2. Uterus and Oviduct. Autoradiographic studies have shown high PBR density in the epithelium and glands of the uterus and lower PBR density in uterine smooth muscle (Verma and Snyder, 1989). In the immature rat, PBR density in the oviduct and uterus increases with age (Fares et al., 1987). In a cycling adult rat, PBR density in both organs increases to a maximal value on the day of proestrus and has been found to correlate with the increase in serum estradiol levels throughout the 4-day cycle (Fares et al., 1988). These data suggest that PBR density in the uterus and oviduct is under hormonal regulation.

3. Adrenal. The highest concentration of PBRs in peripheral tissues is found in the adrenal tissue (De Souza et al., 1985; Fig. 1). Autoradiographic studies have shown PBRs to be present in the adrenal cortex and absent from the adrenal medulla (Benavides et al., 1983a; De Souza et al., 1985). Immunostaining and confocal microscopy on adrenal PBR by Oke et al. (1992) confirmed these findings. The adrenal medulla, however, appears to be enriched with GABA_A receptors that possess PBRs (Kitayama et al., 1989, 1990). The ontogeny of both adrenal mitochondrial PBR binding density and immunoreactivity directly parallels that of the onset of steroidogenesis in the adrenal cortex (Zilz et al., 1999). This is consistent with the notion that PBRs are a prerequisite for adrenocortical steroidogenesis (Zilz et al., 1999).

4. Mammary Gland. Although high PBR density is found in acinar cells in normal mammary gland and in 7,12-dimethylbenz[a]anthracene-induced tumors (Tong et al., 1991), PBR density does not change in response to lactation. Synthesis of DBI has been shown to occur in mammary acinar cells by in situ hybridization (Tong et al., 1991). Whether PBR density in the mammary gland is under hormonal regulation and whether PBRs are involved in mammary cell functions such as casein biosynthesis have yet to be explored.

PBRs have been identified in the testis (Anholt et al., 1985a; De Souza et al., 1985; Mercer et al., 1992), as well as in the vas deferens, prostate, seminal vesicle, and Cowper's gland (Katz et al., 1990a). The hierarchical order of PBR density in the rat genital tract is expressed as follows: testis > Cowper's gland > prostate > vas deferens > seminal vesicle (Katz et al., 1990a). PBRs have been immunolocalized in the rat testis and were found to be present exclusively in the interstitial Leydig cells (Garnier et al., 1993). A 3-fold increase in PBR density was demonstrated in rat testis during maturation (Mercer et al., 1992). These results might reflect critical interactions between PBRs and gonadal hormone activity during development, because testicular PBRs are putatively involved in testosterone production. PBR density in the rat testis is dependent on the trophic influence of pituitary hormones: although hypophysectomy induces depletion of testicular PBR (Anholt et al., 1985a), ligands specific for PBRs increase the in vitro human chorionic gonadotropin-stimulated testosterone secretion of decapsulated rat testis and interstitial cell suspension (Ritta et al., 1987; Ritta and Calandra, 1989).

Treatment with estradiol decreases rat testicular PBR density (Anholt et al., 1985a; Gavish et al., 1986a). The effect obtained with estradiol treatment might be due to its antiandrogenic impact (Gavish et al., 1986a). Cyproterone acetate induces a decrease in rat testicular PBR density (Amiri et al., 1991; Table 3). This effect might be due to its antiandrogenic activity at the target tissue level (Murad and Haynes, 1985). The reduction in rat testicular PBR density after testosterone administration might be due to the suppressive effect of the exogenous androgen on the production of endogenous male hormone via negative feedback (Amiri et al., 1991).

The increase in adrenal PBR density after testosterone administration (Amiri et al., 1991) might be due its anabolic effect, whereas the decrease in adrenal PBR density after cyproterone administration (Amiri et al., 1991) might be due to its antiandrogenic and/or glucocorticoid activity, which suppresses pituitary ACTH secretion and can lead to a loss of adrenal weight (Poyet and Labrie, 1985). The decrease in adrenal PBR density after testicular removal accords with the trend toward diminution proposed by Anholt et al. (1985a). PBR density in the heart was not affected by either cyproterone acetate or testosterone administration (Amiri et al., 1991; Table 3).

Cowper's glands are accessory sex organs that produce the coagulating components of semen and are dependent on the trophic influence of testosterone. Testosterone administration to rats induced an increase in PBR density in Cowper's gland (Amiri et al., 1991; Table 3). PBR density in Cowper's gland was decreased (71%) after testicular removal, whereas testosterone administration prevented this castration-induced PBR depletion (Weizman et al., 1992). PBR depletion in this organ after castration, although prevented by testosterone, indicates that PBRs in this organ are localized on cells whose integrity depends on the trophic influence of testosterone. No significant changes were obtained in PBR density in the heart after testicular removal (Weizman et al., 1992), suggesting that testosterone-related PBR changes may be restricted to the male genital tract.

	VI. Peripheral Benzodiazepine Receptors under Pathological Conditions

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A. Stress Response

1. Relevance of Peripheral Benzodiazepine Receptors to Stress. PBRs seem to be involved in the regulation of several major stress systems: 1) the HPA axis, 2) the sympathetic nervous system, 3) the renin-angiotensin axis, and 4) the neuroendocrine-immune axis. The localization of PBRs on the outer and inner mitochondrial membranes suggests that they may be involved in basic cellular metabolic processes, in addition to their role in the regulation of biosynthesis of steroids. This assumption is supported by the observation of high PBR density in tissues that display high levels of cytochrome oxidase activity and use oxidative phosphorylation for their metabolic needs (Anholt et al., 1986a,b; Hirsch et al., 1989).

2. Peripheral Benzodiazepine Receptor Ligands and Behavior. Behavioral studies have demonstrated that Ro 5-4864 possesses anxiogenic and convulsant properties, whereas PK 11195 has been found to be anxiolytic and anticonvulsant (Weissman et al., 1983, 1984a,b; Bénavidès et al., 1984; Pellow and File, 1984; Mizoule et al., 1985; Massotti and Lucantoni, 1986; Hariton et al., 1988). PK 11195 reverses acute anxiety-induced (forced swimming stress) increase in platelet aggregation, indicating a possible anxiolytic effect of the drug (Serrano et al., 1988). Using Vogel's conflict model of anxiety (thirsty-lick test) in rats, Mizoule et al. (1985) reported that Ro 5-4864 decreased, whereas PK 11195 increased, punished drinking in a dose-dependent fashion. Furthermore, the punishment effects of Ro 5-4864 were antagonized by low doses of PK 11195. These authors showed in this model that Ro 5-4864 and PK 11195 act similarly to CBR inverse agonists and partial agonists, respectively, in a conflict situation. It is of note that neither the Ro 5-4864 nor the PK 11195 behavioral effects were blocked by the CBR antagonist Ro 15-1788, suggesting that the action of Ro 5-4864, at least in Vogel's conflict model of anxiety, is independent of the CBR. Other experimental models support the anxiogenic activity of Ro 5-4864, although some have failed to antagonize this effect with PK 11195 (Pellow and File, 1984; File and Pellow, 1985).

3. Involvement of Peripheral Benzodiazepine Receptors in Acute Stress. a. Animal Studies. The sensitivity of PBRs to acute stress has been demonstrated in various animal models. Drugan et al. (1986) were the first to show the involvement of PBRs in the physiological response to stress, using inescapable tail shocks as an animal model of stress. Five shocks induced a significant increase in the density of renal but not cerebral cortex PBRs. The exposure of mice to acute maximal electroshock induced rapid up-regulation of PBR density in mouse cerebral cortex and cardiac ventricles, measured 30 min after the procedure (Basile et al., 1987).

4. Involvement of Peripheral Benzodiazepine Receptors in Chronic Stress. a. Animal Studies. As described above, exposure of rats to five inescapable tail shocks induced a significant increase in renal PBR density (Drugan et al., 1986). In contrast, 80 repeated tail shocks resulted in a significant decrease of PBR density in kidney, heart, pituitary gland, and cerebral cortex. This biphasic effect of inescapable shock may indicate differences in PBR response to short- versus long-term exposure to stress. This assumption is supported by the finding that repeated swimming stress (daily forced swimming for 21 days) was associated with a significant reduction in renal PBR, in contrast to the up-regulatory effect of single swimming stress on PBR density in this organ (Burgin et al., 1996). Another model demonstrating the association between down-regulation of renal PBR and long-term stress is that of Maudsley reactive rats, which have been bred for high levels of fearfulness. Maudsley reactive rats exhibited decreased renal PBR density compared with Maudsley nonreactive rats (Drugan et al., 1987).

5. Putative Mechanisms Involved in Peripheral Benzodiazepine Receptor Response to Stress. The exact mechanisms that play a role in modulation of PBR expression in response to stress are not well established. PBRs are localized predominantly on the outer mitochondrial membrane, are very dense in peripheral organs that are highly activated during stress (heart, kidney, adrenal, and lung), and have been suggested to be involved in oxidative metabolism and steroidogenesis. Activation of the PBR during acute exposure to a stressor may provide neural and metabolic preparation for better coping with the stress. Furthermore, in some situations, the changes in PBRs are accompanied by a simultaneous increase in CBR activity, a combination that enables efficient psychological and physical fitness (Drugan and Holmes, 1991). Renal PBR activation during acute stress may be relevant to stress-induced hypertension via activation of the renin-angiotensin system (Holmes and Drugan, 1993; Drugan, 1996). In steroidogenic tissues, PBR ligands can affect the translocation of cholesterol from the outer to the inner mitochondrial membrane, although the absolute rate changes are limited (Krueger and Papadopoulos, 1990). Because stress is accompanied by an increase in glucocorticoid synthesis and release, it is possible that this receptor plays a pivotal role in the neuroendocrine response to stress.

B. Anxiety Disorders

1. Generalized Anxiety Disorder. GAD is characterized by excessive anxiety and worry occurring frequently for at least 6 months. The anxiety, worry, or physical symptoms cause clinically significant distress and impairment in social and occupational functioning (American Psychiatric Association, 1994). Reduced (24%) [³H]PK 11195 binding to platelet membranes was observed in GAD patients compared with age- and sex-matched normal controls. Four weeks of diazepam treatment induced a reduction in the anxiety, accompanied by an elevation (69%) in PBR density. One week of diazepam withdrawal resulted in a slight decrease (16%) in PBR density compared with the level during the drug treatment (Weizman et al., 1987b). Similar results are reported in lymphocyte PBRs, and the decrease was restored to a normal value after long-term treatment with BZs (Ferrarese et al., 1990; Rocca et al., 1991). It has been suggested that endogenous neuropeptides such as DBI may be released in anxiety and down-regulate PBR expression (Ferrarese et al., 1990). It is of note that Ro 5-4864 possesses proconflict and anxiogenic effects that can be antagonized by PK 11195 (Mizoule et al., 1985). Furthermore, PK 11195 has been reported to possess anxiolytic properties in psychiatric patients (Papart et al., 1988; Ansseau et al., 1991).

2. Panic Disorder. PD is characterized by recurrent unexpected panic attacks, which lead to persistent concern and worry about having additional attacks and to impairment in daily functioning (American Psychiatric Association, 1994). Platelet PBR density was found to be reduced (29%) in PD patients compared with normal controls (Marazziti et al., 1994), although an earlier study failed to demonstrate such an alteration in lymphocyte membranes (Rocca et al., 1991).

3. Generalized Social Phobia. Patients with GSP have a persistent and excessive fear of one or more social or performance situations in which the person is exposed to unfamiliar people or to possible scrutiny by others. Exposure to the feared social situation provokes anxiety, which may be similar to situationally predisposed panic attack (American Psychiatric Association, 1994). An abnormally low number of platelet PBRs (36%) have been demonstrated in GSP patients (Johnson et al., 1998).

4. Post-Traumatic Stress Disorder (PTSD). PTSD is a severe anxiety disorder that may occur after the exposure to a traumatic event. The traumatic event is persistently reexperienced, and persistent avoidance of stimuli associated with the trauma, numbing of general responsiveness, and symptoms of increased arousal are present (American Psychiatric Association, 1994). Platelet PBRs were assessed in drug-free PTSD patients. All were Israeli citizens who had been exposed to repeated missile attacks during the Persian Gulf War. The PTSD symptoms persisted for 2 years. Civilians from the same geographical area who were exposed to the same attacks composed the control group. Decreased platelet PBR density (62%) was observed in the PTSD patients compared with the control group. The reduction in PBR density in the PTSD patients was more prominent in men than in women (Gavish et al., 1996).

5. Obsessive-Compulsive Disorder (OCD). OCD is characterized by recurrent obsessions and compulsions that cause marked distress, interfere with the patient's daily function, and are commonly associated with depression (American Psychiatric Association, 1994). Two studies have demonstrated a lack of alteration in platelet PBR in OCD patients (Weizman et al., 1993a; Marazziti et al., 1994), whereas one study has demonstrated decreased lymphocyte membrane PBR density (25%) in OCD (Rocca et al., 1991).