Molecular and Cellular Endocrinology, 88 (1992) 1-13 0 1992 Elsevier Scientific Publishers Ireland, Ltd. 0303-7207/92/$0.5.00

MOLCEL 02821

Review

The endocrine system and mitochondrial

benzodiazepine

receptors

Moshe Gavish a,b, Shalom Bar-Arni a,c and Ronit Weizman d a Rappaport

Family Institute for Research in the Medical Sciences and b Department of Pharmacology, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, 31096 Haifa, Israel, ’ Department of Obstetrics and Gynecology, Rambam Medical Center, 31096 Haifa, Israel, and d Tel Aviv Community Mental Health Center and Sackler Faculty of Medicine, Tel Aviv University, 69978 Tel Aviv, Israel

(Accepted 28 May 1992)

Key words: Benzodiazepine

receptors; Endocrine system; Mitochondrion; (Review)

Introduction Central benzodiazepine receptors

Benzodiazepines (BZs) are used clinically as muscle relaxants, anticonvulsants, anxiolytics, and sedative hypnotics. These therapeutic effects are mediated via specific receptors located in the central nervous system (central BZ receptors, CBR) and are coupled with y-aminobutyric acid (GABA) receptors and chloride ion channels (Braestrup and Squires, 1977; Mahler and Okada, 1977; Tallman et al., 1980). The binding of various BZs to these receptors correlates with their clinical potency (Mijhler et al., 1978). CBR have been purified, their complementary DNA (cDNA) cloned, and functional receptors expressed in frog oocytes (Schofield et al., 1987). Mitochondrial BZ receptors

In addition to these receptors, other sites for BZ binding have been identified in various peripheral tissues and in glial cells in the brain. These sites were first referred to as ‘peripheral BZ binding sites’, but since BZs which label these sites have specific effects on various biological systems and since an endogenous ligand for these

Correspondence to: Moshe Gavish, Department of Pharmacology, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, P.O.B. 9649, 31096 Haifa, Israel.

sites has been isolated, they were later called ‘peripheral BZ receptors’ (Verma and Snyder, 1989). These receptors differ from CBR in their lack of coupling to GABA, receptors and their ligand specificity. CBR exhibit high affinity for clonazepam, but not for Ro 5-4864 (4’-chlorodiazepam) or PK 11195 (an isoquinoline carboxamide derivative). The reverse is true for peripheraltype BZ receptors, which exhibit high affinity for Ro 5-4864 and PK 11195, but low affinity for clonazepam. Autoradiographic studies have shown that the pattern of [3H]PK 11195 binding in tissue sections from rat adrenal gland is similar to the histochemical distribution of cytochrome oxidase and monoamine oxidase (MAO) - both of which are markers for mitochondria (Anholt et al., 1986b). Such a correlation has not been found with markers for nuclei, lysosomes, peroxisomes, endoplasmic reticulum, plasma membrane, or cytoplasm. Similar observations have been made in rat testis, lung, kidney, heart, skeletal muscle, liver, and brain (Antkiewicz-Michaluk et al., 1988a): PK 11195 binding essentially parallels the distribution of the mitochondrial enzyme succinate dehydrogenase (Antkiewicz-Michaluk et al., 1988a). Since these receptors have been localized mainly to the mitochondrial membrane, they are now referred to as ‘mitochondrial BZ receptors’ (MBR). Titration of isolated rat mitochondria with digitonin has shown that MBR are released together with MAO, indicating that MBR are

2

associated with the mitochondrial outer membrane (Anholt et al., 1986b). Other studies also support these findings (Mukherjee and Das, 1989; O’Beirne et al., 1990), though it has been reported that MBR can also be localized in nonmitochondrial fractions (Olson et al., 19881. The binding of [“HIPK 11195 and [3HlRo 54864 to various organs has been studied in various species (Basile et al., 1986; Awad and Gavish, 1987,199l; Parola and Laird, 1991). [‘HIPK 11195 binds with high affinity in rat, guinea pig, calf, dog, cat, rabbit, and human; on the other hand, Ro 5-4864 labels rat and guinea pig tissues with high (nanomolar) affinity, but is about 3 orders of magnitude less potent in dog, rabbit, cat, and human tissues and about 4 orders of magnitude less potent in the calf. MBR species differences observed in the membrane-bound state are preserved after solubilization (Awad and Gavish, 1989a), suggesting different structures for MBR in various species rather than primary differences in the membrane environment. Insight into the role of MBR would be facilitated by knowledge of the identity of endogenous ligands. Freezing and thawing membrane preparations of rat kidney decreased the apparent K, of Ro 5-4864 binding to MBR without a change in the B,,, (Gavish and Fares, 1985a), suggesting that physical manipulation may release the endogenous ligand. Red blood cell lysates inhibited ligand binding to MBR; the inhibitory activity was purified and found to be porphyrins (Verma et al., 1987). In this regard, we recently purified a high-molecular-weight heat-stable protein from human cerebrospinal fluid (CSF) that is probably a carrier for porphyrins in the CSF or a general carrier for porphyrins in the blood (Katz et al., 1990a). Endozepines and DBI The demonstration that mitochondrial steroidogenesis is regulated by specific ligands active at the MBR indicates that these receptors may be the target for endogenous ligands which mediate the actions of adrenocorticotropic hormone (ACTH) and luteinizing hormone (LH). Efforts to isolate endogenous ligands for MBR have led to the identification of several naturally occurring substances, including BZ-like compounds, por-

phyrins, and the peptide diazepam-binding inhibitor (DBI) (for review see Costa, 1991, and Costa and Guidotti, 1991). DBI, which is a polypeptide with a molecular weight of 9 kDa, was first isolated in 1983 from rat brain and was shown to displace [3H]diazepam from both CBR and MBR (Costa and Guidotti, 1991). Recently, the DBI gene was localized, cloned, and expressed (Mocchetti and Santi, 1991). In order to differentiate DBI and its processing products from other low-molecular-mass endogenous BZlike compounds, it has been proposed to reserve the term ‘endozepines’ for the endogenous ligands that are immunoreactive with antibodies to BZs, and not to use this term to define DBI and related peptides (Guidotti, 1991). The amount of endozepines present in the brain seems to be biologically significant; however, the biosynthetic pathways of endozepines, their neuronal storage and release mechanisms, and their physiological role are as yet unclear (Olasmaa et al., 1989). Two major naturally occurring DBI-processing products have been identified in rat brain: DBI,,-,, (octadecaneuropeptide, ODN) and DBI ,7_50 (triacontatetraneuropeptide, TTN). Although the structures of the two peptides overlap, they differ in their chemical properties and biological activity profiles. ODN binds preferentially to the GABA, receptor, while TIN binds preferentially to the MBR (for review see Guidotti, 1991). DBI and its processing products regulate steroidogenesis activated by ACTH and LH via binding to MBR, and thus control mitochondrial cholesterol transport (Hall, 1991; Papadopoulos et al., 1991a,b; Brown et al., 1992). The amount of DBI and its rate of synthesis in the adrenal cortex are under the control of ACTH (Massotti et al., 1991). The physiological and pharmacological role of DBI and related peptides in the in vivo regulation of steroidogenesis merits further investigation. Molecular

properties

of MBR

Molecular structure of MBR MBR have been solubilized by digitonin from rat kidney and adrenal gland (Benavides et al., 1985; Gavish and Fares, 1985b1, by Triton X-100

3

from rat kidney (Martini et al., 1983), by sodium cholate from rat kidney (Anholt et al., 1986a), and by CHAPS from cat cerebral cortex (Awad and Gavish, 1989b). MBR have either been partially purified and then photoaffinity labeled by ligands which bind to MBR, or first photoaffinity labeled and then purified. The ligands used for photoaffinity labeling are [3H]PK 14105 [l-(2-fluoro-5-nitrophenyll-3-isoquinoline carboxamidel (Doble et al., 19871, L3H]flunitrazepam (FNZ) (McEnery et al., 1992), and [3HlANH-086 [l-(2isothiocyanatoethyl-7-chloro-1,3-dihydro-5-(4chloro-phenyll-2H-1,4-benzodiazepine-2-one hydrochloride] (McCabe et al., 1989). The first compound is an isoquinoline carboxamide derivative that labels the PK binding site (PKBS), and the other two compounds are BZs. Purification of MBR can aid in understanding their function. MBR have been solubilized by 0.5% dodecyl maltoside from rat mitochondrial kidney and then purified on hydroxylapatite columns (McEnery et al., 1992). Photoaffinity labeling of these partially purified MBR by [ 3HlPK 14105, [3HlFNZ, and [3H]ANH-086, and the use of antibodies against the voltage-dependent anion channel (VDAC) and the adenine nucleotide carrier (ADC), revealed that this receptor is composed of three proteins: binding sites for PK 11195, VDAC, and ADC. These proteins have molecular weights of 18, 32, and 30 kDa respectively. Gel-filtration chromatography of this receptor complex over Superose-12 reveals a single sharp peak with an apparent molecular weight of 70 kDa (McEnery et al., 1992). The l&kDa site is primarily responsible for isoquinoline carboxamide derivative binding, whereas the VDAC and ADC bind BZs. The fact that isoquinolines compete with BZs specific for MBR and that these BZs compete with isoquinoline carboxamide derivative binding to MBR indicates that these sites are closely related (McEnery et al., 1992). Molecular biology of MBR

PKBS have been purified from rat adrenal mitochondria (Antkiewicz-Michaluk et al., 1988b). Rat adrenal mitochondria were first photoaffinity-labeled by [3H]PK 14105, then solubilized by 1% digitonin, and soluble proteins were subjected

to fractionation by ion-exchange and reversephase high-performance liquid chromatography. By the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, radioiodination with Bolton-Hunter reagent, amino acid analysis, gas-phase sequencing, and reverse-phase chromatography, the site for isoquinoline carboxamide has been purified to apparent homogeneity with a molecular weight of 17 kDa. A full-length cDNA comprising 781 base pairs and encoding this 17-kDa protein has been cloned (Sprengel et al., 1989). This cDNA specified an open reading frame of 169 amino acids including the sequences of the PKBS peptide fragments. The PKBS cDNA was injected into an eukaryotic expression vector (containing the SV40 upstream enhancer and the beta globin promoter) which was subsequently transfected into the human kidney 293 cell line for expression of the gene product. Binding studies determined that this gene product binds Ro 5-4864 and PK 11195 with the same specificity as rat MBR. These results indicate that PKBS is a protein essential for the manifestation of MBR. The amino acid sequence of human MBR deduced from corresponding cDNA has been found to be 79% identical to that reported for rat MBR; however, human MBR contain two cysteines, while rat MBR contain none. Finally, with the cDNA for human PKBS as a probe, the PKBS gene has been localized to chromosome 22 (Riond et al., 1991). Involvement of mitochondrial steroidogenesis

BZ receptors

in

Localization of MBR in endocrine organs

Endocrine organs are extremely rich in MBR (Benavides et al., 1983; De Souza et al., 1985). Autoradiography reveals a high density of L3H]Ro 5-4864 binding sites distributed throughout the pituitary, with the highest levels in the posterior lobe (De Souza et al., 1985). In the testis these receptors are localized primarily to the interstitial tissue (Benavides et al., 1983; Calvo et al., 1990), while in the adrenal the receptors are abundant in the cortex and absent in the medulla (Benavides et al., 1983; De Souza et al., 1985). The receptors in the adrenal cortex constitute over 0.2% of the total mitochondrial protein (Antkie-

4

wicz-Michaluk et al., 1988a,b). The presence of MBR has also been demonstrated in human term placenta, a tissue that is also involved in steroidogenesis (Fares and Gavish, 1986). Over the last three years, evidence has emerged that drugs which are active at the MBR affect steroid synthesis in testicular (Ritta and Calandra, 1989), adrenocortical (Besman et al., 1989) and placental (Barnea et al., 1989) tissues. The possibility that MBR are involved in the regulation of endocrine activity of the adrenal gland and testis has been suggested by Anholt et al. (1985), who demonstrated that hypophysectomy induces depletion of MBR in these organs. The reduction in density of the receptors was correlated with the postsurgical atrophy of these tissues. The MBR down-regulation appeared to be a primary effect of the hypophysectomy and indicates that MBR in these endocrine organs are dependent on the trophic influence of the pituitary. This suggestion is further supported by the observation that ACTH administration to hypophysectomized rats increases MBR concentration (Fares et al., 1989). Moreover, steroids affect MBR concentrations in rat testis (Gavish et al., 1986a), and chronic diazepam administration elevates plasma testosterone levels in man (Argiielles and Rosner, 197.5) but not in rats (Wilkinson et al., 1980). A stimulator effect of MBR ligands on steroidogenesis has aIso been obtained in human term placental explants (Barnes et al., 1989) and adrenocortical tissue (Yanagibashi et al., 1989a,b).

MBR ligands and cholesterol transport Various MBR-specific ligands enhance the conversion of cholesterol into pregnenolone (Yanagibashi et al., 1989a,b). Thus, cholesterol conversion into pregnenolone was increased by diazepam in bovine adrenal fasciculata cells (Yanagibashi et al., 1989b) and by both diazepam and Ro 5-4864 in bovine adrenocortical mitochondria (Yanagibashi et al., 1989a). The same group have recently purified a protein from bovine adrenal tissue that stimulated the deiivery of cholesterol to the inner mitochondrial membrane. The primary structure of this protein was almost identical to the primary structure of MBR (Besman et al., 1989). Furthermore, these authors showed that diazepam enhances the delivery of cholestero1 from cell cytoplasm to the inner mitochondrial membrane (Besman et al., 1989). Subsequent studies have demonstrated even more directly that the stimulatory actions of MBR ligands on steroidogenesis are due to increased cholesterol transport to the inner mitochondrial membrane, i.e., MBR mediates transiocation of cholesterol (Krueger and Papadopoulos, 1990).

Role of MBR in steroidogenesis A series of recent studies has elucidated the involvement of specific MBR ligands in the steroid biosynthesis pathway by which MBR mediate the stimulation in steroid hormone production. Pituitary peptide hormones such as ACTH or gonadotropins bind to membranal receptors at their target tissues and activate adenylate cyclase, which subsequently initiates a series of intracellular events mediated by the second messenger adenosine 3,5-cyclic monophosphate (CAMP) (Hall, 1984). The first step in the steroid biosynthesis pathway is the conversion of cholesterol to pregnenolone by cytochrome P-450 side-chaincleavage enzyme (P-450,,,), which is located on the inner mitochondrial membrane (HalI, 1984).

Adrenocortical cells The first step in elucidating the role of MBR in the regulation of steroid production was to investigate the effect of MBR ligands on steroidogenesis in several steroidogenic model systems. Mukhin et al. (1989) have demonstrated that the potencies of MBR ligands to stimulate steroidogenesis in Y-l adren~o~ical cells correlate very closely (r = 0.985) with the potencies to compete with [3H]PK 11195 binding to MBR. These authors suggested that these results prove that MBR ligands regulate steroid production when they bind to the mitochondrial BZ recognition sites. Furthermore, similar effects were observed in the same study in primary cultures of rat and bovine adrenocortical cells.

The transport of cholesterol from the outer to the inner mitochondrial membrane is rate-limiting in steroid production (Krueger and Papadopoulos, 1990). ACTH, LH and follicle-stimulating hormone (FSH) activate this intramitochondrial cholesterol transport in their corresponding target organs (Krueger, 1991).

5

Leydig cells

Other cell models used to study the role of MBR in steroidogenesis are the mouse Leydig cell line MA-10 and purified rat Leydig cells (Papadopoulos et al., 1990). In this study, a high correlation (r = 0.97) was found between steroidogenic potency and the affinity of MBR ligands for their binding sites in the MA-10 Leydig cell line. Stimulation of steroid biosynthesis by human chorionic gonadotropin (hCG), CAMP or MBR ligands is inhibited by the P-450,,, inhibitor aminoglutethimide, indicating that their acute stimulatory effect on steroidogenesis is prior to pregnenolone synthesis. Steroidogenesis induced by hCG and CAMP is inhibited by cycloheximide, a protein synthesis inhibitor which is known to block steroid production at a step prior to conversion of cholesterol to pregnenolone. Cycloheximide does not affect the stimulation of steroidogenesis achieved by MBR ligands (Papadopoulos et al., 19901, indicating that MBR function is at or beyond the outer mitochondrial membrane. The stimulatory effect of MBR ligands has not been observed with mitoplasts (mitochondrial preparation from which the outer mitochondrial membrane is removed), suggesting that they do not act directly on the P-450,,, enzyme (Papadopoulos et al., 1990). It appears that MBR are most likely involved with cholesterol uptake from intracellular stores into mitochondria, promoting cholesterol availability to P-450,,,. These authors suggest that the polypeptide diazepam-binding inhibitor (the endogenous ligand for MBR) which is enriched in steroidogenic tissues may interact physiologically with MBR, thus activating steroidogenesis. A subsequent study demonstrated that FNZ inhibits the actions of the trophic hormones ACTH and hCG on steroid biosynthesis in adrenocortical and Leydig cell lines (Papadopoulos et al., 1991~). FNZ does not completely antagonize the hormone-stimulated steroidogenesis, consistent with its acting as a partial agonist in the absence of any trophic hormone. Antagonistic effect of FNZ

An inhibitory effect of FNZ on pregnenolone production has also been detected in isolated

mitochondrial preparations. These observations indicate that the antagonistic action of FNZ on steroidogenesis is mediated through its interaction with the mitochondrial BZ receptor and demonstrate that hormone-stimulated steroidogenesis is coupled to these recognition sites (Papadopoulos et al., 1991~1. Granulosa cell steroidogenesis

The effect of MBR-specific ligands on granulosa cell steroidogenesis has been tested in primary granulosa cells and in a granulosa cell line transformed by SV40 T-antigen and Ha-rus oncogene (Amsterdam and Suh, 1991). In both cell types, Ro 5-4864 and Ro 5-2807 produced 3- to 5-fold stimulation of progesterone secretion, whereas the CBR-specific ligands Ro 15-4513 and Ro 15-1788 were ineffective. Interestingly, there is evidence that in short-term incubation (2 h) addition of either forskolin, an effector of adenylate cyclase, or FSH fails to enhance the effect of Ro 5-4864 on the progesterone secretion from primary granulosa cells or a transformed granulosa cell line. Furthermore, the effect of MBRspecific ligands was significantly higher in granulosa cells cultured in cholesterol-deficient medium than in medium supplemented with cholesterol derived from fetal calf serum. Interaction ous organs

between hormones

and MBR in vari-

Pineal gland

Suranyi-Cadotte et al. (1987) have demonstrated the presence of MBR in the human pineal gland. This gland and its hormone melatonin may play a role in sleep promotion, regulation of biological rhythms, and affective disorders. These functions may be related to an interaction with central and peripheral BZ receptors in the pineal gland. However, the functional role of MBR in this gland is as yet unclear. In the rat, pineal MBR have been found to be associated with catecholamine nerve terminals (Quirion, 1984; Weissman et al., 1984). BZs augment norepinephrine stimulation of melatonin synthesis in rat pineal gland via MBR (Matthew et al., 1981). Melatonin can reduce anxiety-like behavior in animals; thus, it is possible that pineal MBR are

6

involved in neurophysiological response to stress (Suranyi-Cadotte et al., 1987). Thyroid gland

Chronic thyroid hormone treatment in rats results in the up-regulation of MBR in heart, kidney and testis (Gavish et al., 1986b). The modulatory effect of thyroxine on MBR expression may be related to a compensatory adaptation mechanism to the hyperthyroid state associated with hypermetabolism and stress. Studies on the effects of MBR ligands on the symptoms of thyrotoxicosis, and of experimental hyperthyroidism on these receptors, may clarify the interaction between MBR and thyroid hormones. Kidney

The kidney contains a high density of MBR, especially in the ascending limb of the loop of Henle, the distal convoluted tubule and collecting tubules (Beaumont et al., 1984). High rates of sodium and chloride transport and water reabsorption occur in these regions, which are sensitive to the hormones aldosterone and vasopressin (Edelman, 1981). Alterations in renal MBR density have been reported in progesterone-treated rats (Gavish et al., 19871, uninephrectomized hypertensive rats treated with deoxycorticosterone acetate and salt (Regan et al., 1981>, spontaneously hypertensive rats (Taniguchi et al., 1981) and in an animal model of diabetes insipidus, the vasopressin-deficient Brattleboro rat (Del Zompo et al., 1983). Adrenalectomy reduces MBR density in the rat kidney (Basile et al., 1985), and this reduction is reversed by administration of aldosterone. The reduction is confined to the cortical layers of the kidney and may reflect trophic changes in the cells comprising the distal segments of the nephron in response to mineralocorticoids (Basile et al., 1987). This line of evidence suggests that MBR in the kidney may play an important role in anion transport in the kidney. Male genital tract

Autoradiography reveals a high density of MBR in the interstitial tissues of the testis (De Souza et al., 1985). Apart from the testis, MBR have been identified and characterized in the vas

deferens, prostate, seminal vesicle, and Cowper’s gland (Katz et al., 1990b). In the rat testis, MBR are dependent on the trophic influence of pituitary hormones, and hypophysectomy causes a decrease in MBR density in the testis (Anholt et al., 1985). MBR ligands increase the in vitro and hCG-stimulated testosterone secretion of decapsulated testis (Ritta et al., 1987; Ritta and Calandra, 1989). These observations are in accordance with a report of stimulatory effect of chronic diazepam treatment in male patients on testosterone secretion (Argiielles and Rosner, 1975). Chronic testosterone treatment in male rats induces a decrease in MBR concentration in the testis and an increase in that in Cowper’s gland, while administration of the antiandrogenic agent cyproterone acetate does not affect MBR density in Cowper’s gland and selectively depletes testicular MBR (Amiri et al., 1991). The reduction in testicular MBR concentration following testosterone administration may reflect the suppressive effect of the exogenous androgen on the production of pituitary gonadotrophins via negative feedback. Cowper’s gland, like the prostate and seminal vesicle, is dependent upon the trophic influence of testosterone, and testosterone administration is thus followed by a significant increase in MBR density in Cowper’s gland. It is possible that the antiandrogenic activity of cyproterone acetate is counteracted by the progestational and glucocorticoid properties of this agent, preserving MBR levels in Cowper’s gland. Removal of the testis induces a reduction of MBR density in Cowper’s gland, and testosterone administration prevents the castration-induced depletion (Weizman et al., 1992). Thus, it seems that MBR in Cowper’s gland are localized on cells the integrity of which depends on the trophic influence of testosterone, whereas the testicular MBR, which are localized on Leydig cells, are gonadotrophin dependent. Ovary

MBR have been localized in the rat ovary (Fares et al., 1987). Higher MBR concentrations have been detected in granulosa cells, and interstitial theta cells from immature rats treated with pregnant mare serum gonadotropin (PMSG) (Bar-Ann, Fares and Gavish, unpublished data).

7

MBR have also been detected in granulosa cells from Chinese hamster (Riond et al., 1989) and human (Bar-Ami et al., 1991) ovary, as well as in rat granulosa cell lines transformed by SV40 Tantigen and Ha-rus oncogene (Amsterdam and Suh, 1991). In the immature rat ovary, MBR density increases with age (Fares et al., 1987). Hypophysectomy prevents this age-dependent increase and causes a significant decrease in ovarian MBR density associated with follicular atresia and a decrease in ovarian weight (Bar-Ann et al., 1989). The effect of hypophysectomy can be abolished either by 2 days treatment with PMSG or 4 days treatment with the synthetic estrogen diethylstilbestrol (DES) (Bar-Ami et al., 1989). In intact rat, PMSG or DES also significantly increases MBR density (Fares et al., 1987). Short-term (4 days) treatment with either progesterone or testosterone increases MBR density in the ovary. However, long-term (10 days) treatment results in a significant decrease in MBR density (Bar-Ami, Amiri, Fares and Gavish, submitted for publication). In the adult rat, ovarian MBR density increases to a maximum on the day of proestrus (Fares et al., 1988). On the day of proestrus, follicular size is maximal and the follicle is ready to ovulate; maximal MBR concentrations are already seen on the night following diestrus, i.e., 14 h before the onset of the LH surge (Fares et al., 1988). In rabbit ovary, however, although MBR have been demonstrated in interstitial theta cells, they have been found in granulosa cells only following ovulation at the onset of luteinization (Verma and Snyder, 1989). Granulosa cells

In granulosa cells obtained from Chinese hamster ovary, MBR have been identified as a protein of the mitochondrial outer membrane (Riond et al., 1989). Using the photoaffinity probe 13H]PK 14105, a nitrophenyl derivative of [3H]PK 11195, these authors have purified a 17-kDa mitochondrial protein which presumably contained at least part of the MBR recognition site (Riond et al., 1989). Also, in granulosa cell lines transformed by SV40 T-antigen and Ha-ras oncogene, [3H]Ro 5-4864 demonstrated saturable binding to the iso-

lated mitochondria. This specific binding could be displaced by specific peripheral MBR ligands such as unlabeled Ro 5-4864 or Ro 5-2807, but could not be displaced by specific CBR ligands such as Ro 15-4513 or Ro 15-1788 (Amsterdam and Suh, 1991). In these in vitro conditions a significant increase in MBR concentration was observed in response to various agents which increase intercellular CAMP level (Amsterdam and Suh, 1991). MBR are thus detectable in ovarian homogenates, hamster and rat granulosa cells, and human and rabbit granulosa-lutein cells. Further studies have demonstrated that MBR are localized in the mitochondrial outer membrane, and that MBR concentrations in both in vitro and in vivo studies are hormone dependent. Data on the hormonal regulation of MBR density suggest that MBR concentrations in the ovary increase with granulosa cell differentiation, which is associated with in vivo follicular growth and development. The association between degree of differentiation and MBR concentrations in the ovarian cells of various rodents encouraged us to study MBR density in the human ovary. High MBR concentrations were demonstrated in human granulosalutein cells obtained at in vitro fertilization/embryo transfer (IVF/ET). It appears that MBR concentrations are significantly greater in granulosa-lutein cells obtained from larger follicles or when morphological luteinization of follicular cells has been observed (Bar-Arm et al., 1991). Furthermore, when MBR concentrations were studied in individual follicles, a high correlation was found between egg cell performance fin terms of completion of meiotic maturation to the second metaphase stage, fertilization and embryonic cleavage) and MBR-specific binding in the corresponding granulosa cells (Bar-Ami et al., 1991). MBR concentrations in human ovarian granulosa cells were significantly higher in women with high plasma estradiol-17P levels ( > 1400 pg/ml) compared with women with lower levels (< 1000 pg/ml). Moreover, granulosa cell MBR concentrations were l.&fold higher in women who conceived following IVF/ET treatment than in those who did not conceive (Bar-Arm et al., 1991). The effect of BZ ligands on the steroidogenic activity of human granulosa cells obtained from

8

women subjected to hormonally induced ovulation and luteinization has been studied. PK 11195, as well as Ro 5-4864, caused an approximately 2-fold increase in in vitro secretion (P < 0.02). Furthermore, the stimulatory effect of these ligands upon granulosa cell progesterone secretion coincided with the increase in [3H]PK 11195 maximal binding (B,,,) capacity, which may suggest a receptor-mediated action (Bar-Ann et al., unpublished data). Thus, in the ovary, MBR concentration is under endocrine, paracrine and autocrine regulation, and MBR-specific ligands also appear to modulate ovarian follicular cell steroidogenic activity.

Uterus and oviduct

Autoradiographic studies have revealed high MBR concentrations localized in the uterine endometrium and gland, but lower MBR concentrations in uterine smooth muscle (Verma and Snyder, 1989). MBR concentrations in the oviduct and uterus of the immature rat increase with age (Fares et al., 1987), and hypophysectomy results in a significant decrease in both organs (Bar-Ami et al., 1989). Furthermore, MBR concentrations in uterus and oviduct of the cycling adult rat increase to a maximal value on the day of proestrus (Fares et al., 1988) further evidence that MBR concentrations in these organs are under hormonal regulation. MBR concentrations increased significantly in the oviduct and uterus of intact immature rats treated with PMSG for 2 days (Fares et al., 1987); such treatment also reversed the dramatic decrease in uterine and oviduct MBR concentrations observed in hypophysectomized rats (BarAmi et al., 1989). PMSG contains both FSH and LH activity (Moor and Ward, 1980), and acts directly on the ovary to induce multiple follicular growth and development in the immature rat, and also elicits a significant increase in ovarian estradiol biosynthesis (Braw and Tsafriri, 1980). Thus, the inductive effect of PMSG treatment on MBR concentration could be mediated by PMSG stimulation of ovarian estradiol biosynthesis. This interpretation is supported by studies on the administration of DES to intact (Fares et al., 1987) or hypophysectomized (Bar-Ann et al., 1989) rats,

in which significantly increased MBR concentrations in the oviduct and uterus were seen. Short-term (4 days) treatment with testosterone but not progesterone increased MBR concentrations in the oviduct and uterus; long-term (10 days) treatment with testosterone or progesterone caused a > 35% reduction in the oviduct. Short-term but not long-term administration of testosterone was associated with significant increases in serum estradiol levels. Thus, we may suggest that the increase in MBR concentrations in DES and in short-term testosterone treatment could be at least partially mediated by estrogenic action on the uterus and oviduct. In cycling adult rats, MBR concentrations in each individual uterus have been found to correlate with the corresponding serum estradiol levels throughout the 4-day cycle (Fares et al., 1988). MBR concentrations in the uterus were significantly reduced by long-term treatment with testosterone (approximately 25% reduction), but long-term treatment with progesterone was ineffective (Bar-Ami, Amiri, Fares and Gavish, submitted for publication). The reduction in MBR could be associated with the inhibiting effect of testosterone on ovarian follicular growth usually observed with high testosterone dosage or following long-term treatment with this steroid (Louvett et al., 1975). Recently we have studied changes in MBR concentration in samples of human uterine endometrium collected on different days of the menstrual cycle. PK 11195 showed a specific and saturable binding to the uterine endometrium, on Scatchard analysis to a single population of sites. Lower MBR concentrations were found at the time of menses and in the early and late secretory-luteal phase. In comparison with these phases, a 3- to 4-fold increase in MBR concentration was seen in the follicular proliferation phase, just before or at the time of ovulation, and in the middle secretory phase (Bar-Ann, Erlick, Amiri, Fares, Blumenfeld and Gavish, submitted for publication). The increase in MBR concentration in the uterine endometrium coincided with an increase in serum estradiol level, further evidence for the critical role of estradiol in the regulation of MBR in female genital organs. Estradiol, which increases MBR concentrations in the oviduct and uterine endometrium, is

9

also involved in developmental processes in these organs such as an increase in the glandular epithelium (Branham et al., 1985). Some of these developmental processes are inhibited or stimulated by MBR-specific ligands. Estradiol-induced ciliogenesis in the oviduct is inhibited by BZ ligands such as diazepam and medazepam, which also inhibit the beating of mature cilia (BoisvieuxUlrich et al., 1987). Furthermore, testing the effect of MBR-specific ligands on oxytocin-induced contractions in rat uterine tissue revealed that diazepam and Ro 5-4864, but not clonazepam, inhibit amplitude of uterine contractions by 50% (Katz et al., 1990a). Collectively, in the oviduct and uterus MBR-specific ligands exert an inhibitory effect on a variety of developmental and functional activities of these organs. Recently the interaction between MBR and the GABA, receptor has been studied in human myometrium. Thus, [3H]FNZ binding to membrane preparations was enhanced in the presence of GABA, and [ 14C]GA13A binding was enhanced in the presence of FNZ (Sergeev et al., 1990). In the human, the spontaneous contractile activity of the ampullar segment of the Fallopian tube can be increased by some GABAergic compounds (Lsszl et al., 1990). Given that GABA-induced contraction of rat uterine muscle is inhibited by progesterone or progesterone metabolites (Putnam et al., 19911, the effects of these agents on the regulation of oviduct and uterine contraction should be further studied against the possibility of interactions between GABA, MBRspecific ligands and steroids in peripheral as well as central sites. Mammary gland

High MBR concentrations are detectable in acinar cells, both in normal mammary gland and 7,lZdimethylbenz[a]anthracene-induced tumors (Tony et al., 1991). Lactation is not associated with a change in MBR localization. Synthesis of a diazepam-binding inhibitor has been shown to occur in mammary acinar cells by in situ hybridization (Tony et al., 1991). Whether MBR concentrations in the mammary gland are under hormonal regulation, and whether MBR are involved in mammary cell functions such as casein biosynthesis, have yet to be explored.

Adrenal

The highest concentration of MBR in peripheral tissues is found in the adrenal, whether whole tissue (De Souza et al., 1985) or isolated mitochondria (Antkiewicz-Michaluk et al., 1988a). Autoradiographic studies have shown MBR in the adrenal cortex and absent from the adrenal medulla (Benavides et al., 1983; De Souza et al., 1985), whereas the adrenal medulla is enriched with CBR, which are coupled to the GABA, receptor (Kitayama et al., 1989). Nevertheless, in the PC12 pheochromocytoma line, which is a noradrenergic clonal line of rat adrenal medullary tumor cells, high MBR density has been induced by forskolin and nerve growth factor (Miller et al., 1988b). Removal of the pituitary gland, thereby eliminating ACTH secretion, causes a significant reduction in adrenal MBR density (Anholt et al., 1985); similarly, indirect interference with ACTH secretion affects MBR density in the adrenal. Thus, adrenal MBR density is significantly reduced in male rats treated with cyproterone (Amiri et al., 19911, an antiandrogenic drug, probably as a consequence of the reduction in pituitary ACTH secretion caused by this drug (Poyet and Labrie, 1985). The effect of hypophysectomy on the reduction in MBR density is manifested mainly in the zona fasciculata and zona reticularis, whereas no effect has been noted in the zona glomerulosa (Anholt et al., 1985). Since hypophysectomy causes a significant reduction in both ACTH level and adrenal glucocorticoid biosynthesis and secretion, mineralocorticoid- and glucocorticoid-dependent MBR (such as in the kidney) are significantly reduced following hypophysectomy (Anholt et al., 1985) or adrenalectomy (Basile et al., 1985). Fares et al. (1989) demonstrated that in hypophysectomized rats, ACTH administration increased MBR concentrations in both the adrenal and the kidney; however, hydrocortisone administration increased concentrations only in the kidney. Various MBR-specific ligands may alter steroidogenic activity in the adrenal cortex. The effect of these ligands can be seen over the entire hypothalamic-pituitary-adrenal axis. Thus, Ro 5-4864 directly stimulates the release of corticotropin-releasing hormone (CRH) but not

10

ACTH, whereas PK 11195 directly stimulates the secretion of ACTH (Calogero et al., 19901, and in early studies diazepam was reported to increase plasma corticosterone (Marc and Marselli, 1969). This latter observation has been disputed in more recent studies; in various models of induced stress in rats, the increase in corticosterone and ACTH was attenuated or abolished by treatment with diazepam (Copland and Balfour, 1987). Also, in human subjects suffering from panic or generalized anxiety disorder, treatment with diazepam caused a significant reduction in ACTH and cortisol (Roy-Byrne et al., 1991). A partial answer to these contradictory observations may be attributable to diazepam, which has the capacity to bind to both CBR and MBR (Gobbi et al., 1987). Thus, alprazolam, a CBRspecific ligand, is capable of suppressing the hypothalamic-pituitary-adrenal axis in primates, which most likely reflects suppression of CRH neurons rather than the pituitary corticotroph (Kalogeras et al., 1990). Taken together, it is possible that MBR-specific ligands stimulate adrenal cortical steroidogenic activity by acting at a number of loci in the hypothalamic-pituitary axis, and that CBR-specific ligands produce the opposite effect by suppression of the hypothalamic secretion of CRH. In adrenal glomerulosa cell culture, addition of various MBR-specific ligands increased angiotensin II-induced aldosterone secretion. The effect of these various ligands coincides with the rank of their potential to displace L3HlPK 11195 from these cells, which suggests a receptor-mediated action (Song and Zhou, 1989). However, in potassium-induced (but not in forskolin-induced) aldosterone secretion addition of diazepam has been reported to produce an inhibitory effect (Shibata et al., 1986). Various BZ ligands may alter other indices of adrenal cortical activity. Thus, in male Wistar rats following monoadrenalectomy diazepam administration reduces the mitotic index of the adrenal cortex (Zieleniewski et al., 1990a,b). This effect of BZs is not surprising, since various BZs have been demonstrated to reduce peripheral cell proliferation and induce cell differentiation (Clark et al., 1965; Matthew et al., 1981; Wang et al., 1984). In the pituitary, however, the MBR ligand Ro

5-4864, but not the CBR ligand Ro 15-1788, enhanced [ 3H]thymidine incorporation (Stepien et al., 1986). Many studies have shown that various MBRspecific ligands enhance adrenal cell steroidogenie activity by increasing cholesterol translocation from the intercellular stores to the mitochondrial P-450,,, (Besman et al., 1989; Muhkin et al., 1989). Recently Holloway et al. (1989) have shown that the CBR-specific ligand midazolam and the mixed MBR/CBR ligand diazepam inhibit cortisol and aldosterone synthesis in bovine adrenal cells in vitro. The inhibitory effect of diazepam on potassium-induced aldosterone secretion by adrenal glomerulosa cells has also been reported (Shibata et al., 1986). Midazolam was a more potent inhibitor than diazepam, with each affecting 17a- and 21-hydroxylation (Holloway et al., 1989). Nevertheless, the concentration needed to achieve this effect is significantly higher than the therapeutic levels. Adrenal steroids have been shown to regulate BZ receptors in mice (Miller et al., 1988a) and rat (Acuiia et al., 1990). Also, ACTH has been found to alter [ 3H]GABA, receptor binding in rat brain (Kendall et al., 1982). Thus, in rats, hypophysectomy or adrenalectomy causes a significant increase in cerebral cortex [ 3H]FNZ binding. Corticosterone administration returned B,,, to normal value in adrenalectomized and hypophysectomized rats (Acuiia et al., 1990). Concluding

overview

BZs are used clinically as muscle relaxants, anticonvulsants, anxiolytics, and sedative hypnotics and act through the GABA,/BZ receptor complex located in the central nervous system. In addition to these CBR, there is another type of receptor, which also binds BZs but is located on the mitochondria of peripheral organs and on the glial cells in the brain - the MBR. MBR differ from CBR in their drug specificity and lack of coupling to GABA, receptors and the chloride ion channel. Recent studies indicate that MBR are composed of three subunits: isoquinoline binding site, voltage-dependent anion channel, and adenine nucleotide carrier, with molecular weights of 18, 32, and 30 kDa, respectively. The

11

site for isoquinoline has been purified from rat adrenal mitochondria; a full-length cDNA comprising 781 base pairs and encoding the site for isoquinoline has been cloned. The amino acid sequence of human MBR deduced from corresponding cDNA has been found to be 79% identical to that reported for rat MBR. Since the initial discovery of Braestrup and Squires (1977) that BZs bind with high affinity and specificity to non-neuronal tissues, numerous studies have been employed to study the MBR in many tissues and organs of various species. The biochemical characteristics and physiological significance of the MBR have been defined. The major role of MBR is in the regulation of steroid biosynthesis. ACTH, LH and FSH activate via MBR intramitochondrial cholesterol transport in their corresponding endocrine target organs. Various MBR-specific ligands enhance the conversion of cholesterol into pregnenolone and the production of steroid hormones. The stimulatory effect of MBR-specific ligands on pregnenolone production is inhibited by FNZ. The naturally occurring peptide DBI and its processing products stimulate in vivo steroidogenesis via activation of MBR. The expression of MBR is under the control of the pituitary hormones ACTH, FSH and LH as well as the peripheral steroid hormones. MBR are also involved in uterine and oviduct contractions_ At this stage the molecular structure of MBR, their endogenous ligands, and the functions of these receptors have been elucidated. However, the signaling system activated by MBR awaits further investigation. Acknowledgement

We thank Miss Ruth Singer for typing and editing the manuscript. References Acuiia, D., Fernandez, B., Gomar, M.D., del Aguila, M. and Castillo, J.L. (1990) Neuroendocrinology 51, 97-103. Amiri, Z., Weizman, R., Katz, Y., Burstein, O., Edoute, Y., Lochner, A. and Gavish, M. (1991) Brain Res. 553, 155158. Amsterdam, A. and Suh, B.S. (1991) Endocrinology 129, 503510.

Anholt, R.R.H., De Souza, E.B., Kuhar, M.J. and Snyder, S.H. (1985) Eur. J. Pharmacol. 110, 41-46. Anholt, R.R.H., Aebi, U., Pedersen, P.L. and Snyder, S.H. (1986a) Biochemistry 25, 2120-2125. Anholt, R.R.H., Pedersen, P.L., De Souza, E.B. and Snyder, S.H. (1986b) J. Biol. Chem. 261, 576-583. Antkiewicz-Michaluk, L., Guidotti, A. and Krueger, K.E. (1988a) Mol. Pharmacol. 34, 272-278. Antkiewicz-Michaluk, L., Mukhin, A.G., Guidotti, A. and Krueger, K.E. (1988b) J. Biol. Chem. 263, 17317-17321. Argiielles, A.E. and Rosner, J. (1975) Lancet ii, 607. Awad, M. and Gavish, M. (1987) J. Neurochem. 49,1407-1414. Awad, M. and Gavish, M. (1989a) Biochem. Pharmacol. 38, 3843-3849. Awad, M. and Gavish, M. (1989b) J. Neurochem. 52, 18801885. Awad, M. and Gavish, M. (1991) Life Sci. 49, 1155-1161. Bar-Ami, S., Fares, F. and Gavish, M. (1989) Horm. Metab. Res. 21, 106-107. Bar-Ami, S., Fares, F. and Gavish, M. (1991) Mol. Cell. Endocrinol. 82, 285-291. Barnea, E.R., Fares, F. and Gavish, M. (1989) Mol. Cell. Endocrinol. 64, 155-159. Basile, AS., Paul, S.M. and Skolnick, P. (1985) Eur. J. Pharmacol. 110, 149-150. Basile, A.S., Klein, D.C. and Skolnick, P. (1986) Mol. Brain Res. 1, 127-135. Basile, AS., Ostrowski, N.L. and Skolnick, P. (1987) J. Pharmacol. Exp. Ther. 240, 1006-1013. Beaumont, K., Healy, D.P. and Fanestil, D.D. (1984) Am. J. Physiol. 247, F718-F724. Benavides, J., Malgouris, C., Imbault, F., Begassat, F., Uzan, A., Renault, C., Dubroeucq, M.C., Gueremy, C. and Le Fur, G. (1983) Arch. Int. Pharmacodyn. Ther. 266, 38-49. Benavides, J., Menager, J., Burgevin, M.C., Ferris, O., Uzan, A., Gueremy, C., Renault, C. and Le Fur, G. (1985) Biochem. Pharmacol. 34, 167-170. Besman, M.J., Yanagibashi, K., Lee, T.D., Kawamura, M., Hall, P.F. and Shiveley, J.E. (1989) Proc. Natl. Acad. Sci. USA 86, 4897-4901. Boisvieux-Ulrich, E., Laine, MC. and Sandoz, D. (1987) Cell Motil. Cytoskeleton 8, 333-344. Braestrup, C. and Squires, R. (1977) Proc. Nat]. Acad. Sci. USA 79, 3805-3809. Branham, W.S., Sheehan, D.N., Zehr, D.R., Ridlon, E.R. and Nelson, C.J. (1985) Endocrinology 117, 2229-2237. Braw, R.H. and Tsafriri, A. (1980) J. Reprod. Fertil. 59, 259-265. Brown, A.S., Hall, P.F., Shoyab, M. and Papadopoulos, V. (1992) Mol. Cell. Endocrinol. 83, 1-9. Calogero, A.E., Kmilaris, T.C., Bernardini, R., Johnson, E.O., Chrousos, G.P. and Gold, P.W. (1990) J. Pharmacol. Exp. Ther. 253, 729-737. Calve, D.J., Ritta, M.N., Calandra, R.S. and Medina, J.H. (1990) Neuroendocrinology 52, 350-353. Copland, A.M. and Balfour, D.J. (1987) Pharmacol. Biochem. Behav. 27, 619-624. Costa, E. (1991) Neuropharmacology 30, 1357-1364.

12 Costa, E. and Guidotti, A. (1991) Life Sci. 49, 325-349. De Souza, E.B., Anholt, R.R.H., Murphy, K.M.M., Snyder, S.H. and Kuhar, M.J. (1985) Endocrinology 116, 567-573. Del Zompo, M., Bocchetta, A., Corsini, G.U., Tallman, J.F. and Gessa, G.L. (19831 in Benzodiazepine Recognition Site Ligands: Biochemistry and Pharmacology (Biggio, G. and Costa, E., eds.1, pp. 239-248, Raven Press, New York. Doble, A., Ferris, O., Burgevin, M.C., Menager, J., Uzan, A., Dubroeucq, M.C., Renault, C., Gueremy, C. and Le Fur, G. (1987) Mol. Pharmacol. 31, 42-49. Edelman, I.S. (1981) Am. J. Physiol. 241, F333-F339. Fares, F. and Gavish, M. (1986) Biochem. Pharmacol. 35, 227-230. Fares, F., Bar-Ann, S., Brandes, J.M. and Gavish, M. (1987) Eur. J. Pharmacol. 133, 97-102. Fares, F., Bar-Ann, S., Brandes, J.M. and Gavish, M. (1988) J. Reprod. Fertil. 83, 619-625. Fares, F., Bar-Ami, S., Haj-Yehia, Y. and Gavish, M. (1989) J. Receptor Res. 9, 143-157. Gavish, M. and Fares, F. (1985a) Eur. J. Pharmacol. 107, 283-284. Gavish, M. and Fares, F. (1985b) J. Neurosci. 5, 2889-2893. Gavish, M., Okun, F., Weizman, A. and Youdim, M.B.H. (1986a) Eur. J. Pharmacol. 127, 147-151. Gavish, M., Weizman, A., Okun, F. and Youdim, M.B.H. (1986b) J. Neurochem. 47, 1106-1110. Gavish, M., Weizman, A., Youdim, M.B.H. and Okun, F. (1987) Brain Res. 409, 386-390. Gobbi, M., Barone, D., Mennini, T. and Garattini, S. (1987) J. Pharm. Pharmacol. 39, 388-391. Guidotti, A. (1991) Neuropharmacology 30, 1425-1433. Hall, P.F. (1984) Int. Rev. Cytol. 86, 53-95. Hall, P.F. (1991) Nenropharmacology 30, 1411-1416. Holloway, CD., Kenyon, C.J., Dowie, L.J., Corrie, J.E.T., Gray, C.E. and Fraser, R. (1989) J. Steroid Biochem. 33, 219-225. Kalogeras, K.T., Calogero, A.E., Kuribayiashi, T., Khan, I., Gallucci, W.T., Kling, M.A., Chrousos, G.P. and Gold, P.W. (1990) J. Clin. Endocrinol. Metab. 70, 1462-1471. Katz, Y., Allon, N., Abraham, S., Oz, N. and Gavish, M. (1990a) in Calcium Channel Modulators in Heart and Smooth Muscle: Basic Mechanisms and Pharmacological Aspects (Abraham, S. and Amitai, G., eds.), pp. 253-268, VCH, Weinheim and Balaban, Rehovot. Katz, Y., Amiri, Z., Weizman, A. and Gavish, M. (1990b) Biochem. Pharmacol. 40, 817-820. Kendall, D.A., McEwen, B.S. and Enna, S.J. (1982) Brain Res. 236, 365-374. Kitayama, S., Morita, K., Dohi, T. and Tsujimoto, A. (1989) Arch. Int. Pharmacodyn. Ther. 300, 254-264. Krueger, K.E. (1991) Neuropsychopharmacology 4, 237-244. Krueger, K.E. and Papadopoulos, V. (1990) J. Biol. Chem. 265, 15015-15022. Lgszl, O.A., NHdasy, G.L., ErdG, S.L., Monos, E., Skili%.i, G. and Zsolnai, B. (1990) Acta Physiol. Hung. 76, 123-130. Louvett, J.P., Harman, S.M., Schrieber, J.R. and Ross, G.T. (1975) Endocrinology 97, 366-372.

Marc, L. and Marselli, P.L. (1969) J. Pharm. Pharmacol. 21, 784-789. Martini, L., Giannaccini, G. and Lucadini, A. (1983) Biochim. Biophys. Acta 728, 289-292. Massotti, M., Slobodyansky, E., Konkel, D., Costa, E. and Guidotti, A. (1991) Endocrinology 129, 591-596. Matthew, E., Parfitt, A.G., Sugden, D., Engelhardt, D.L., Zimmerman, A. and Klein, D.C. (1981) J. Pharmacol. Exp. Ther. 228, 434-438. McCabe, R.J., Schoenheimer, J.A., Skolnick, P., Newman, A.H., Rice, K.C., Reig, J.-A. and Klein, D.C. (1989) FEBS Lett. 244, 263-267. McEnery, M.W., Snowman, A.M., Trifiletti, R.R. and Snyder, S.H. (1992) Proc. Natl. Acad. Sci. USA 89, 3170-3174. Mider, L.G., Greenblatt, D.J., Barnhill, J.G., Tompson, M.L. and Shader, L. (1988a) Brain Res. 446,314-320. Miller, L.G., Tischler, A.S., Jumblatt, J.E. and Greenblatt, D.J. (1988b) Neurosci. Len. 89, 342-348. Mocchetti, I. and Santi, M.R. (1991) Neuropharmacology 30, 1365-1371. Mohler, H. and Okada, T. (1977) Science 198, 849-851. Mohler, H., Okada, T., Heitz, P.H. and Ulrich, J. (1978) Life Sci. 22, 985-996. Moor, W.T. and Ward, D.N. (1980) J. Biol. Chem. 255, 6930-6936. Mukherjee, S. and Das, S.K. (1989) J. Biol. Chem. 264, 16713-16718. Mukhin, A.G., Papadopoulos, V., Costa, E. and Krueger, K.E. (1989) Proc. Natl. Acad. Sci. USA 86, 9815-9816. O’Beirne, G.B., Woods, M.J. and Williams, D.C. (1990) Eur. J. Biochem. 188, 131-138. Olasmaa, M., Guidotti, A., Costa, E., Rothstein, J.D., Goldman M.E., Weber, RJ. and PauI SM. (1989) Lancet i, 491-492. Olson, J.M.M., Ciliax, B.J., Mancini, W.R. and Young, A.B. (1988) Eur. J. Pharmacol. 152, 47-53. Papadopoulos, V., Mukhin, A.G., Costa, E. and Krueger, K.E. (19901 J. Biol. Chem. 265, 3772-3779. Papadopoulos, V., Berkovich, A. and Krueger, K.E. (1991a) Neuropharmacology 30, 1417-1423. Papadopoulos, V., Berkovich, A., Krueger, K.E. and Costa, E. (1991b) Endocrinology 129, 1481-1488. Papadopoulos, V., Nowzari, F.B. and Krueger, K.E. (1991~) J. Biol. Chem. 266, 3682-3687. Parola, A.L. and Laird, H.E. (1991) Life Sci. 48, 757-764. Poyet, P. and Labrie, F. (1985) Mol. Cell. Endocrinol. 42, 283-288. Putnam, C.D., Brann, D.W., Kolbeck, R.C. and Mahesh, V.B. (1991) Biol. Reprod. 45, 266-272. Quirion, R. (1984) Eur. J. Pharmacol. 102, 559-560. Regan, J.W., Yamamura, H.I., Yamada, S. and Roeske, W.R. (1981) Life Sci. 28, 991-998. Riond, J., Vita, N., Le Fur, G. and Ferrara, P. (1989) FEBS Lett. 245, 238-244. Riond, J., Mattei, M.G., Kaghad, M., Dumont, X., Guillemot, J.C., Le Fur, G., Caput, D. and Ferrara, P. (1991) Eur. J. Biochem. 195, 305-311.

13 Ritta, M.N. and Calandra, R.S. (1989) Neuroendocrinology 49, 262-266. Ritta, M.N., Campos, M.B., Campos, R.S. and Calandra, R.S. (1987) Life Sci. 40, 791-798. Roy-Byrne, P.P., Cowley, D.S., Hommer, D., Ritchie, J., Greenblatt, D. and Nemeroff, C. (1991) Biol. Psychiatry 30, 73-80. Schofield, P.R., Darlison, M.G., Fujita, N., Burt, D.R., Stephenson, F.A., Rodriguez, H., Rhee, L.M., Ramachandran, J., Reale, V., Glencorse, T.A., Seeburg, P.H. and Barnard, E.A. (1987) Nature 328, 221-227. Sergeev, P.V., Sizov, PI., Dukhanin, A.S. and Mineeva, E.N. (1990) Bull. Exp. Biol. Med. (Engl. Transl. Byull. Eksp. Biol. Med.) 110, 382-384. Shibata, H., Kojima, I. and Ogata, E. (1986) Biochem. Biophys. Res. Commun. 135, 994-999. Song, L.C. and Zhou, T.C. (1989) Life Sci. Early Sci. 32, 458-467. Sprengel, R., Werner, P., Seebury, P.H., Mukhin, A.G., Santi, M.R., Grayson, D.R., Guidotti, A. and Krueger, K.E. (1989) J. Biol. Chem. 264, 20415-20421. Stepien, H., Kunert-Radek, J. and Pawlikowski, M. (1986) J. Neural Transm. 66, 303-307. Suranyi-Cadotte, B., Lal, S., Nair, N.P.V., Lafaille, F. and Quirion, R. (1987) Life Sci. 40, 1537-1543. Tallman, J.F., Paul, S.M., Skolnick, P. and Gallager, D.W. (1980) Science 207, 279-281.

Taniguchi, T., Wang, J.K.T. and Spector, S. (1981) Eur. J. Pharmacol. 70,587-588. Tony, Y., Rheaume, E., Simard, J. and Pelletier, G. (1991) Regul. Pept. 33, 263-273. Verma, A. and Snyder, S.H. (1989) Annu. Rev. Pharmacol. Toxicol. 29, 307-322. Verma, A., Nye, J.S. and Snyder, S.H. (1987) Proc. Natl. Acad. Sci. USA 84, 2256-2260. Weissman, B.A., Skolnick, P. and Klein, D.C. (1984) Pharmacol. Biochem. Behav. 21, 821-824. Weizman, A., Amiri, Z., Katz, Y., Snyder, S.H. and Gavish, M. (1992) Brain Res. 572, 72-75. Wilkinson, M., Moger, W. and Grovestine, D. (1980) Life Sci. 27, 2285-2291. Yanagibashi, K., Ohno, Y., Nakamichi, N., Matsui, T., Hayashida, K., Takamura, M., Yamada, K., Tou, S. and Kawamura, M. (1989a) J. Biochem. 106, 1026-1029. Yanagibashi, K., Ohno, Y., Nakamichi, N., Matsui, T., Hayashida, K., Takamura, M., Yamada, K., Tou, S. and Kawamura, M. (1989b) Jpn. J. Pharmacol. 51, 347-355. Zieleniewski, J., Nowakowska-Jankiewicz, B. and Stepieh, H. (1990a) Cytobios 61, 85-88. Zieleniewski, J., Nowakowska-Jankiewicz, B., Stepieh, H. and Zieleniewski, W. (199Ob) Cytobios 64, 81-85.