0013-7227/91/1292-059l$03.00/0 Endocrinology Copyright ^ 1991 by The Endocrine Society

Vol. 129, No. 2 Printed in U.S.A.

Regulation of Diazepam Binding Inhibitor in Rat Adrenal Gland by Adrenocorticotropin M. MASSOTTI*, E. SLOBODYANSKY, D. KONKEL, E. COSTA, AND A. GUIDOTTI Fidia-Georgetown Institute for the Neurosciences, Georgetown University School of Medicine, Washington, D.C. 20007

glands decreased dramatically from approximately 80 to 15 ng/ mg tissue. The administration of single dose of ACTH (ACTH residues 1-39; 200 mU/kg, iv) or repeated doses of ACTH-R (ACTH in saline containing 16% gelatin; 15 U/kg, sc, twice daily) reduced the decrease in adrenal DBI-LI caused by hypophysectomy. In hypophysectomized rats (7 days after hypophysectomy) the increases in both adrenal DBI-LI and plasma corticosterone induced by ACTH 1 h after a single injection (200 mU/kg, iv) were inhibited by injection of cycloheximide (40 mg/ kg, ip) 10 min after ACTH. However, cycloheximide at this dose had no effect on the ACTH-induced increase in adrenal cAMP concentration or the number or affinity of MBRs for 4'-[3H] chlorodiazepam. {Endocrinology 129: 591-596, 1991)

ABSTRACT. Diazepam binding inhibitor (DBI) is a 9-kDa polypeptide that was initially isolated from rat brain and subsequently found to be present in several peripheral tissues. DBI is particularly abundant in steroidogenic tissues, such as the adrenal glands and testes, which also contain a high concentration of peripheral/mitochondrial benzodiazepine receptors (MBRs). Because occupancy of adrenal MBRs with DBI results in increased steroidogenesis, we have investigated the relation between ACTH, DBI, and the MBR in the rat adrenal glands. Evidence presented here indicates that both the amount of DBI and its rate of synthesis in the adrenal cortex are under the control of ACTH. Seven and 9 days after hypophysectomy, the amount of DBI-like immunoreactivity (DBI-LI) in rat adrenal

D

IAZEPAM binding inhibitor (DBI) was initially isolated from rat brain by its ability to displace [aH]diazepam from its binding sites on neuronal membranes (1). DBI is a 9-kDa precursor (2-4) of a newly discovered family of neuropeptides that act in brain at the benzodiazepine (BZD) recognition site associated with the type A 7-aminobutyric acid receptor (GABAA receptor) and at the so-called mitochondrial BZD receptor (MBR) (5-8). The presence of DBI in tissues other than brain (2-4, 9-12) suggests that this polypeptide might also be an endogenous ligand for MBRs located on select populations of mitochondria in peripheral tissues (3, 11, 12). Immunocytochemical and biochemical studies have shown that in the periphery, DBI and DBIlike peptides are highly concentrated in the cells of the zona glomerulosa fasciculata and reticularis of adrenal cortex (12,13) and in Leydig cells (12,14). Interestingly, these cell types are highly enriched in MBRs (15), and it has been shown that MBR stimulation with appropriate ligands results in the mobilization of cholesterol into the inner mitochondrial membrane, where cytochrome

P450 catalyzes its transformation into pregnenolone and thereby promotes steroidogenesis (16, 17). Because DBI displaces 4'-[3H]chlorodiazepam from MBRs located on the outer mitochondrial membrane of adrenal cortical cells (12), it has been suggested that DBI may serve as an endogenous modulator of steroidogenesis through its action at the MBR (12, 13). Indeed, this hypothesis has received further support from the observation that incubation of adrenocortical and Leydig cell mitochondria with purified rat brain DBI results in stimulation of pregnenolone biosynthesis (18). It has been known for some years that the stimulation of steroid synthesis in adrenal cortex by ACTH and cAMP requires the de nouo synthesis of one or more proteins that facilitate cholesterol transport into mitochondria and thereby stimulate the synthesis of pregnenolone, from which other adrenal steroids are formed (1924). Recent studies have suggested that Des-[Gly-Ile] DBI is the physiological messenger that mediates the ACTH-induced stimulation of steroidogenesis in the adrenal gland (13). This information prompted us to determine the effect of ACTH on the concentration of DBI in the adrenal cortex of hypophysectomized rats in order to establish whether DBI synthesis is under ACTH control and whether the ACTH-induced release of corticosteroids is associated with changes in the concentration of DBI in the adrenal cortex.

Received February 1,1991. Address all correspondence and requests for reprints to: Dr. A. Guidotti, Fidia-Georgetown Institute for the Neurosciences, Georgetown University School of Medicine, 3900 Reservoir Road NW, Washington, D.C.20007. * Present address: Laboratory of Pharmacology, Istituto Superiore di Sanita, Rome, Italy 00161.

591

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 18:18 For personal use only. No other uses without permission. . All rights reserved.

ACTH REGULATION OF ADRENAL DBI

592

Materials and Methods Animals Intact, sham-operated, and hypophysectomized male Sprague-Dawley rats (200-250 g) were purchased from Zivic-Miller (Allison-Park, PA). The animals were housed in groups of five per cage, supplied with food and water ad libitum, and maintained on a 12-h light, 12-h dark cycle. Animal care and use were in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals (U.S. DHHS, 1985). Drugs Human ACTH (residues 1-39) was purchased from ParkeDavis (Morris Plains, NJ). Cycloheximide was purchased from Sigma (St. Louis, MO) and dissolved in concentrated acetic acid, after which the solution was adjusted to pH 4 with 1 M NaOH. Experimental procedures All experiments were performed between 0900-1200 h in order to minimize the effects of circadian changes in ACTH production in intact and sham-operated animals. When indicated, either sham-operated or hypophysectomized rats, at different time intervals (1-9 days) after surgery, received a single injection of saline (1.0 ml/kg, iv), ACTH (200 mU/kg, iv) diluted in saline, cycloheximide (40 mg/kg, ip), or ACTH followed by cycloheximide. Cycloheximide was injected 10 min after saline or ACTH, and the animals were killed 50 min later. In time-course studies, the effect of ACTH was assessed 30, 60, 120, and 240 min after the injection. In a separate series of experiments, either sham-operated or hypophysectomized rats were treated twice per day (0800 and 2000 h) for several days (as indicated) with saline containing 16% gelatin (1 ml/kg, sc, vehicle) or ACTH [15 U/kg, diluted in 16% gelatin (ACTHR)] in order to provide a prolonged release of ACTH after the injection; for these experiments, pharmacological tests were performed or animals were killed 12 h after the last injection. Sample collection Animals were killed with a guillotine. The adrenal glands (decapsulated), testes, and cerebellum were immediately frozen on dry ice and then stored at -70 C until the day of the assay. For several experiments involving the assay of DBI in cerebellum, rats were killed with a 10-kwatt microwave beam (New Japan Radio, Tokyo, Japan) focused on the animal's head for 1 sec. Because similar results were obtained with animals killed by microwave and those killed by guillotine, separate subgroups are not presented. DBI RIA The frozen tissue from each animal was homogenized for 20 sec in 1 M acetic acid (20 vol for adrenal glands and 10 vol for other tissues) using an Ultra-Turrax homogenizer (Tekmar, Cincinnati, OH). The homogenate was adjusted to pH 5 by the addition of 10 M NaOH. The suspension was centrifuged at 48,000 x g for 20 min at 4 C. The pellet was discarded, and the clear supernatant was diluted 10-, 25-, 50-, and 100-fold with 50 mM sodium phosphate buffer (pH 7.2) containing 5% BSA.

Endo-1991 Vol 129* No 2

Two 50-/ul portions of each dilution were assayed for DBI by RIA, which was performed as previously described (25). The immunological characteristics and specificity of the antisera to DBI have also been previously described (26). cAMP RIA The soluble acetic acid extract of adrenal used in the DBI assay was diluted 10-, 25-, 50-, and 100-fold with 50 mM sodium acetate buffer (pH 5.8), and two 50-^1 portions of each dilution were assayed for cAMP with a RIA kit from Amersham (Arlington Heights, IL). Plasma corticosterone RIA Blood samples were collected from the trunk immediately after decapitation. Heparin (100 U) was added to 1 ml blood, which was then centrifuged at 900 x g for 15 min at room temperature. Corticosterone was assayed in plasma with a RIA kit from ICN (Costa Mesa, CA). Assay of MBR with 4'-(*H]chlorodiazepam (Ro5-4864) The binding of 4'-[3H]chlorodiazepam to crude adrenal homogenates was determined as described previously (27); diazepam (5 ixM) was used as a displacing agent. 4'-[3H]Chlorodiazepam was used at a concentration of 1 nM, which corresponds to the linear range of the binding curve (the dissociation constant, Ka, under our conditions was approximately 2 nM). Protein assay Protein was assayed according to the calorimetric method of Lowry et al. (28). Statistical analysis The statistical significance of the differences between the results from various treatments was determined by Student's t test. In several cases the data were processed through a twoway analysis of variance.

Results Effect of hypophysectomy on DBI-like immunoreactiuity (DBI-LI) in the adrenal glands, testes, and cerebellum and on the plasma corticosterone concentration The amount of DBI-LI in the adrenal glands and testes as well as the weight and protein content of these organs were significantly lower in male rats 9 days after hypophysectomy than in sham-operated controls (Table 1). No significant effect of hypophysectomy on these three parameters was observed in the cerebellum (Table 1). When the amount of DBI-LI was expressed per mg tissue or per mg protein, however, only the adrenals showed a significant decrease in DBI-LI content; DBI-LI in testes was decreased only marginally (Table 1). The plasma concentration of corticosterone decreased from 240 ± 70 to 12 ± 4.2 ng/ml (n = 6; P < 0.001) 9 days after hypophysectomy. The time course of the decrease in the amount DBI-LI in the adrenals after hypophysectomy is

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 18:18 For personal use only. No other uses without permission. . All rights reserved.

ACTH REGULATION OF ADRENAL DBI

593

TABLE 1. DBI-LI content, protein content, and weight of adrenals, testes, and cerebellum of sham-operated (SO) and hypophysectomized (Hpx) male rats 9 days after surgery Treatment

Organ Adrenals

A) B) C) D)

SO + vehicle SO + ACTH-R Hpx + vehicle Hpx + ACTH-R

Testes

A) B) C) D)

SO + vehicle SO + ACTH-R Hpx + vehicle Hpx + ACTH-R

Cerebellum

A) B) C) D)

SO + vehicle SO + ACTH-R Hpx -1- vehicle Hpx + ACTH-R

Organ DBI-LI (ng) 3.6 ± 0.30 4.1 ± 0.38° 0.13 ± 0.012* 2.3 ± 0.25c 190 ± 20 210 ± 25 68 ± 10* 74 ± 8.5* 43 ± 40 ± 38 ± 35 ±

5.0 6.8 4.0 3.9

Organ wt (mg)

Organ protein (mg)

42 ± 2.0 59 ± 7.2° 8.9 ± 1.2* 39 ± 2.5C

4.7 ± 0.32 7.1 ± 0.66" 1.1 ±0.15* 4.0 ± 0.34c

86 ± 70 ± 15 ± 58 ±

1300 ± 120 1500 ± 210 600 ± 104* 555 ± 32*

72 ± 5.0 76 ±11 35 ±4.1* 37 ±7*

143 ± 136 ± 112 ± 130 ±

18 25 19 15

32 ± 29 ± 29 ± 28 ±

140 ± 140 ± 120 ± 130 ±

12 20 10 13

300 ± 280 ± 270 ± 265 ±

12 21 18 14

DBI-LI (ng/mg tissue)

4.0 3.8 2.5 3.0

7.5 8.0 1.6* 6.0OC

Animals received ACTH-R (15 U/kg, sc) or vehicle twice daily from days 3-8 after surgery and were killed 12 h after the last injection. Each value is the mean ± SE for at least six rats. " P < 0.05 compared with SO plus vehicle. b P< 0.001 compared with SO plus vehicle. c P < 0.001 compared with Hpx plus vehicle.

100 200 -i

E m

Q

Control

Days After Surgery FIG. 1. Effect of hypophysectomy on the DBI-LI content of adrenal glands. The adrenal DBI-LI content of hypophysectomized (§1) and sham-operated (•) rats was measured at various times after surgery. Control animals had not undergone surgery and were killed together with the group of rats killed 1 day after surgery. Each value is the mean ± SE for at least eight rats. *, P < 0.05; **, P < 0.01 (compared with sham-operated rats).

shown in Fig. 1. The decrease was already significant on day 1 (18%) and reached a maximum (~80%) on day 7 after surgery. Reversal of the hypophysectomy-induced decrease in adrenal DBI-LI by ACTH treatment A single iv injection of ACTH administered 1 or 9 days after hypophysectomy increased adrenal DBI-LI (Fig. 2). The amount of adrenal DBI-LI remained unchanged 5 and 30 min after the injection of ACTH (200 mU/kg),

Control Min After ACTH

30

60

120

240

(200 mUnits/kg, j.v.)

FIG. 2. Effect of a single injection of ACTH on adrenal DBI-LI content in hypophysectomized rats. The amount of DBI-LI in adrenal glands of male rats that were hypophysectomized 1 day (•) or 9 days (M) before the experiment was determined at different times after a single injection of saline (control) or ACTH (200 mU/kg, iv). The control value (100%) refers to the adrenal DBI-LI content in saline-treated rats and corresponds to 60 ± 4.0 and 15 ± 0.9 ng/mg tissue 1 and 9 days after hypophysectomy, respectively. These values did not change with time after injection of saline. For the ACTH-treated rats, each value represents the mean ± SE for at least five rats. **, P < 0.01 compared with control rats.

but increased approximately 2-fold by 1 h after the injection and remained elevated for up to 4 h. The percent increase in DBI-LI induced by ACTH was approximately the same both 1 and 9 days after hypophysectomy (Fig. 2), even though the basal amount of adrenal DBI-LI was significantly greater in animals 1 day

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 18:18 For personal use only. No other uses without permission. . All rights reserved.

ACTH REGULATION OF ADRENAL DBI

594

Endo•1991 Vol 129 «No 2

after hypophysectomy (Fig. 1). The effect of ACTH on DBI-LI appeared to be specific for the adrenal; there was no effect of ACTH on the amount of DBI-LI in testes or cerebellum, either 1 or 4 h after the ACTH injection. The effects of repeated injections of ACTH-R (ACTHR applied twice daily from days 3-8 after surgery) on adrenal DBI-LI in hypophysectomized and sham-operated rats 9 days after surgery are shown in Table 1. Repeated and protracted administration of ACTH (the last injection was administered 12 h before the animals were killed) reversed the decrease in adrenal DBI-LI caused by hypophysectomy (compare groups C and D for adrenals in Table 1), but failed to change the amount of adrenal DBI-LI in sham-operated animals (group B for adrenals in Table 1). The repeated ACTH treatment also restored the weight of the adrenal gland (Table 1) to values close to those in sham-operated animals, but failed to change the weight, protein content, or DBI-LI content of testes and cerebellum (Table 1).

mized 7 days earlier was not affected by the cycloheximide injection (Table 2). In the same rats, ACTH and cycloheximide failed to change the content of MBRs and their affinity for 4'-chlorodiazepam (Table 2). In hypophysectomized rats, the extent of 4'-[3H]chlorodiazepam binding was approximately one third that measured in sham-operated rats (Table 2). In an attempt to elucidate the role of adrenal DBI in steroidogenesis, the plasma corticosterone concentration was measured in the same group of hypophysectomized rats (Table 2). In general, there was a significant correlation (r = 0.830; P < 0.01) between the amount of adrenal DBI-LI and the plasma corticosterone concentration. Moreover, cycloheximide, which prevented the acute increase in adrenal DBI-LI elicited by a single iv injection of ACTH, also prevented the increase in plasma corticosterone induced by the same treatment (Table 2).

Cycloheximide blocks the increases in adrenal DBI-LI and plasma corticosterone induced by ACTH

DBI is abundant in the zona reticularis and fasciculata of the adrenal gland (12, 13), and it has been suggested that this peptide acts as an intracellular mediator of ACTH-induced steroidogenesis (13,29). Our results demonstrate that both the DBI concentration and the de nouo synthesis of this peptide in adrenal cortex are under the control of ACTH released from the pituitary gland. The progressive decrease in adrenal DBI-LI that follows hypophysectomy was promptly reversed by the administration of ACTH. Although there was also a reduction in adrenal weight and protein content in male rats after hypophysectomy, the decrease in the amount of DBI-LI was much faster and greater. It is possible that the small amount of DBI-LI (~15%) remaining in the adrenals 7 and 9 days after hypophysectomy may actually be stored in adrenal cells [adrenal medulla cells (12)] that are not

The increase in adrenal DBI-LI 1 h after a single injection of ACTH (200 mU/kg, iv) administered 7 days after hypophysectomy was completely inhibited by cycloheximide treatment (40 mg/kg, ip, administered 10 min after ACTH, Table 2). Similarly, cycloheximide blocked the ACTH-induced increase in adrenal DBI-LI observed 7 days after hypophysectomy in rats that had also been supplemented with twice daily injections of ACTH-R from days 4-6 after surgery (Table 2). The amount of adrenal DBI-LI in rats that received saline instead of the acute challenge with ACTH was not modified by cycloheximide. Interestingly, the increase in adrenal cAMP concentration 1 h after ACTH injection to rats hypophysecto-

Discussion

TABLE 2. Effect of ACTH and cycloheximide (Cyclo) on the adrenal content of DBI, cAMP, and MBR and the plasma corticosterone concentration in rats 7 days after hypophysectomy (Hpx) Plasma corticosterone (ng/ml)

Adrenal Acute treatment

DBI-LI (ng/mg tissue) Hpx

Control ACTH Cyclo ACTH + Cyclo

14 ± 26 ± 16 ± 15 ±

1.7 2.5 " 0.7 2.5

Hpx + ACTH-R

cAMP (pmol/mg tissue) Hpx

45 ± 4.0 82 ± 3.6" 48 ± 4.5 43 ± 6.0

0.80 ± 0.05 1.20 ± 0.10* 0.84 ± 0.06 1.25 ± 0.095*

MBR (pmol/mg protein)0 Hpx 1.8 ± 1.6 ± 2.0 ± 1.8 ±

0.10 0.13 0.08 0.12

Hpx

Hpx + ACTH-R

15 ± 2.5 125 ± 10c 42 ± 5.2* 25 ± 8.0

35 ±6.5 780 ±100 c 50 ±9.5 32 ±10

The (Hpx + ACTH-R) group received twice daily injections of ACTH-R (15 U/kg, sc) from day 4 to day 6 after surgery. Animals receiving acute ACTH treatment (200 mU/kg, iv) were injected 1 h before they were killed. Cycloheximide (40 mg/kg, ip) was injected 10 min after ACTH. Each value is the mean ± SE for five to seven rats. 0 4'-[3H]Chlorodiazepam binding in adrenals of sham-operated rats was 5.5 ± 0.32 pmol/mg protein. * P < 0.01 compared with the respective controls. c P < 0.001 compared with the respective controls.

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 18:18 For personal use only. No other uses without permission. . All rights reserved.

ACTH REGULATION OF ADRENAL DBI under the direct control of ACTH. In testis, another steroidogenic organ sensitive to hypophysectomy, the concentration of DBI was independent of ACTH control. In this organ, DBI-LI is present in Leydig cells, Sertoli cells, and probably other cell types (12, 14). The total amount of testicular DBI-LI was reduced after hypophysectomy; however, the concomitant reduction in the size of the organ masked the virtual disappearance of DBI-LI from Leydig cells (30) that has been described after hypophysectomy. Single or repeated injections of ACTH, which reversed the decline in adrenal cortical DBI-LI, failed to modify the amount of DBILI in testes in either control or hypophysectomized animals. A similar independence from ACTH control could explain the stability of cerebellar DBI-LI after hypophysectomy. The regulation of adrenal DBI by ACTH coupled with the observations that DBI displaces BZD receptor ligands from the MBR (12) and that bovine adrenal Des[Gly-Ile]DBI (13) and rat brain DBI (18) increase the conversion of cholesterol to pregnenolone in adrenal mitochondria offer support to the view (13, 29) that DBI is an important intracellular mediator of the steroidogenic action of ACTH in the adrenal cortex. It is well established that in order to induce steroidogenesis in adrenal cortical cells, ACTH first binds to cell surface receptors and activates adenylate cyclase (31); cAMP produced by this enzyme then initiates a complex series of intracellular events, in which proteins with a short half-life are required to mediate the transfer of cholesterol to the cytochrome P450 side-chain cleavage enzyme present intramitochondrially (19-24). In our experiments with cycloheximide a significant positive correlation was found between the amount of adrenal DBI-LI and the plasma corticosterone concentration in hypophysectomized rats that received either vehicle or ACTH. Neither the increase in cAMP induced by ACTH (an index of ACTH receptor function) nor the number or affinity of 4'-[3H]chlorodiazepam-binding sites (at which DBI presumably acts) appeared to be acutely modified by ACTH-cycloheximide treatment. Moreover, in vitro studies (17, 18) have indicated that cycloheximide does not prevent the DBI-elicited conversion of cholesterol to pregnenolone in isolated adrenal mitochondria. These findings are, thus, consistent with the view (13, 29) that de nouo synthesis of DBI in the adrenal could be an important factor in the mediation of ACTH-induced steroidogenesis. However, a closer inspection of the time course of changes in the adrenal DBI concentration after ACTH administration indicates that changes in the DBI concentration cannot be directly related to ACTH-induced steroidogenesis. In hypophysectomized rats, the peak increase in plasma corticosterone after a single ACTH

595

injection occurs within 10-30 min and precedes the increase in DBI concentration that occurs 2-4 h later, when the effect of ACTH on plasma corticosterone concentration is fading. The half-life of DBI in rat adrenal after hypophysectomy appears to be several hours and is probably not very different from the half-life of steroidogenic enzymes, such as cytochrome P450-dependent steroid hydroxylase (32), or of the MBR (27) after hypophysectomy. Thus, the temporal correlation between DBI content and corticosteroid levels during ACTH treatment of hypophysectomized rats does suggest a relation between DBI and the long term effect of ACTH on glucocorticosteroid secretion. In contrast, because of the discrepancies in time courses, the blockade by cycloheximide of the ACTH-induced increases in the adrenal DBI concentration and the plasma corticosterone concentration is not sufficient to establish a cause-effect relation between de novo synthesis of DBI and short term stimulation of steroidogenesis and to conclude that DBI is the cycloheximide-sensitive protein with a short half-life that has been identified as the physiological modulator of cholesterol transport to the inner mitochondrial membrane (29). However, it is still possible that a short-lived activated state of DBI may be important in promoting ACTH-induced short term steroidogenesis. Although no data are presently available on the dynamic changes and intracellular compartmentalization of DBI in adrenal cortical cells, one may speculate that the early phase of ACTH-induced steroidogenesis may be sustained by a small pool of DBI or its processing products that became activated (for example, by phosphorylation) and readily available to specific mitochondrial receptors. Whereas we have previously shown that DBI is not a good substrate for phosphorylation (15), we are accumulating evidence that in the adrenal cortex, DBI can be rapidly processed to several smaller fragments. In an independent series of experiments, it has been demonstrated that DBI-processing products (that is DBI residues 17-50) are more potent and efficacious than DBI itself in stimulating steroidogenesis (33). Therefore, it is possible that ACTH-induced changes in the rate of DBI processing, rather than in the concentration of DBI itself, are responsible for ACTH-induced steroidogenesis. It remains to be ascertained whether ACTH, acting through cAMP-dependent protein kinase, can change the activity of the enzymes (exo- and endopeptidases) that cleave DBI. It also remains to be established whether ACTH can increase steroidogenesis in the absence of changes in DBI or its processing products by acting at the level of MBRs; for example, by changing the affinity of MBRs by the phosphorylation of these receptors.

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 18:18 For personal use only. No other uses without permission. . All rights reserved.

ACTH REGULATION OF ADRENAL DBI

596 References

1. Guidotti A, Forchetti CM, Corda MG, Konkel D, Bennett CD, Costa E 1983 Isolation, characterization and purification to homogeneity of an endogenous polypeptide with agonistic action on benzodiazepine receptors. Proc Natl Acad Sci USA 80:3531-3533 2. Mocchetti I, Einstein R, Brosius J 1986 Putative diazepam binding inhibitor peptide: cDNA clones from rat. Proc Natl Acad Sci 83:7221-7225 3. Gray PW, Glaister D, Seeburg PH, Guidotti A, Costa E 1986 Cloning and expression of a cDNA for human diazepam binding inhibitor, a natural ligand of an allosteric regulatory site of the gamma-aminobutyric acid type A receptor. Proc Natl Acad Sci USA 83:7547-7551 4. Webb NR, Rose TM, Malik N, Marquardt H, Shoyab M, Todaro GJ, Lee DC 1987 Bovine and human cDNA sequences encoding a putative benzodiazepine receptor ligand. DNA 6:71-79 5. Ferrero P, Santi MR, Conti-Tronconi B, Costa E, Guidotti A 1986 Study of an octadecaneuropeptide derived from diazepam binding inhibitor (DBI): biological activity and presence in rat brain. Proc Natl Acad Sci USA 83:827-831 6. Slobodyansky E, Guidotti A, Wambebe C, Berkovich A, Costa E 1989 Isolation and characterization of a triakontatetraneuropeptide (TTN) a posttranslational product of diazepam binding inhibitor: specific action at Ro 5-4864 recognition sites. J Neurochem 53:1276-1284 7. Berkovich A, McPhie P, Compagnone M, Guidotti A, Hensley P 1990 A natural processing product of rat diazepam binding inhibitor, triakontatetraneuropeptide (diazepam binding inhibitor 1750) contains an a-helix, which allows discrimination between benzodiazepine binding site subtypes. Mol Pharmacol 37:164-172 8. Bender AS, Hertz L 1986 Octadeaceneuropeptide (ODN; anxiety peptide) displaces diazepam more potently form astrocytic than from neuronal binding sites. Eur J Pharmacol 132:335-336 9. Ball JA, Burnet PW, Fountain BA, Ghatei MA, Bloom SR 1986 Octadecaeneuropeptide, benzodiazepine ligand-like immunoractivity in rat central nervous system, plasma and peripheral tissues. Neurosci Lett 72:183-188 10. Owens GP, Sinha AK, Sikela JM, Hahn WE 1989 Sequence and expression of the murine diazepam binding inhibitor. Mol Brain Res 6:101-108 11. Alho H, Fremeau RT, Tiedge H, Wilcox J, Bovolin P, Brosius J, Roberts JL, Costa E 1988 Diazepam binding inhibitor gene expression: location in brain and peripheral tissues of rat. Proc Natl Acad Sci USA 85:7018-7022 12. Bovolin P, Schlichting JL, Miyata M, Ferrarese C, Guidotti A, Alho H 1990 Distribution and characterization of diazepam binding inhibitor (DBI) in peripheral tissues of rat. Regul Peptides 29:267281 13. Besman MJ, Yanagibashi K, Lee TD, Kawamura M, Hall PF, Shively JE 1989 Identification of des-(Gly-Ile) endozepine as an effector of corticotropin-dependent adrenal steroidogenesis: stimulation of cholesterol delivery is mediated by peripheral benzodiazepine receptor. Proc Natl Acad Sci USA 86:4897-4901 14. Rheaume E, Tonon MC, Smih F, Simard J, Desy L, Vaudry H, Pelletier G 1990 Location of the endogenous benzodiazepine ligand octadecaneuropeptide in the rat testes. Endocrinology 127:19861994 15. De Souza EB, Anholt RR, Murphy KM, Snyder SH, Kuhar MJ 1985 Peripheral-type benzodiazepine receptors in endocrine organs: autoradiographic localization in rat pituitary, adrenal and testes. Endocrinology 116:567-573

Endo«1991 Vol 129 • No 2

16. Papadopoulos V, Mukhin AG, Costa E, Krueger KE 1990 The peripheral-type benzodiazepine receptor is functionally linked to Leydig cell steroidogenesis. J Biol Chem 265:3772-3779 17. Krueger KE, Papadopoulos V 1990 Peripheral-type benzodiazepine receptors mediate translocation of cholesterol from outer to inner mitochondrial membranes in adrenocortical cells. J Biol Chem 265:15015-15022 18. Papadopoulos V, Berkovich A, Krueger KE, The role of diazepam binding inhibitor and its processing products at mitochondrial benzodiazepine receptors: stimulation of steroid biosynthesis. Neuropharmacology, in press 19. Fergeson Jr JJ 1963 Protein synthesis and adrenocorticotropin responsiveness. J Biol Chem 238:2754-2759 20. Farese RV 1964 Changes in [14C]glycine-incorporating activities of rat-adrenal microsomes and soluble cell fraction during prolonged adrenocorticotropin adminstration. Biochim Biophys Acta 87:515521 21. Garren LD, Ney RL, Davis WW 1965 Studies on the role of protein synthesis in the regulation of corticosterone production by adrenocorticotropic hormone in vivo. Proc Natl Acad Sci USA 53:14431450 22. Simpson ER, McCarthy JL, Petersen JA 1978 Evidence that the cycloheximide-sensitive site of adrenocorticotropic hormone action is in mitochondrion. J Biol Chem 253:3135-3139 23. Privalle CT, Crivello JF, Jeffcoate CR 1983 Regulation of intramitochondrial cholesterol transfer to site-chain cleavage P-450 in rat adrenal gland. Proc Natl Acad Sci USA 80:702-706 24. Connely DM, Headon DR, Olson CO, Ungar F, Dempse ME 1984 Intramitochondrial movement of adrenal sterol carrier protein with cholesterol in response to corticotropin. Proc Natl Acad Sci USA 81:2970-2974 25. Ferrarese C, Vaccarino F, Alho H, Mellstrom B, Costa E, Guidotti A 1987 Subcellular location and neuronal release of diazepam binding inhibitor. J Neurochem 48:1073-1102 26. Alho H, Costa E, Ferrero P, Fujimoto M, Cosenza-Murphy D, Guidotti A 1985 Diazepam binding inhibitor: a neuropeptide located in selected neuronal populations of rat brain. Science 229:179-182 27. Anholt RR, De Souza EB, Kuhar MJ, Snyder SH 1985 Depletion of peripheral-type benzodiazepine receptors after hypophysectomy in rat adrenal gland and testes. Eur J Pharmacol 110:41-46 28. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ 1951 Protein measurement with the Folin phenol reagent. J Biol Chem 193:265275 29. Yanagibashi K, Ohno Y, Kawamura M, Hall PF 1988 The regulation of intracellular transport of cholesterol in the bovine adrenal cells: purification of a novel protein. Endocrinology 123:2075-2082 30. Alho H, Expression of DBI in central nervous system and peripheral organs: the possible role as endogenous regulator of different types of benzodiazepine receptors. Neuropharmacology, in press 31. Grahame Smith DG, Butcher RW, Ney RL, Sutherland EW 1967 Adenosine 3',5'-monophosphate as the intracellular mediator of the action of adrenocorticotropic hormone on the adrenal cortex. J Biol Chem 242:5535-5541 32. Purvis JL, Canick JA, Mason JI, Estabrook RW, McCarthy JL 1973 Lifetime of adrena cytochrome P450 as influenced by ACTH. Ann NY Acad Sci 212:319-343 33. Papadopoulos V, Berkovich A, Krueger KE, Costa E, Guidotti A, Diazepam binding inhibitor (DBI) and its processing products stimulate mitochondrial steroid biosynthesis via an interaction with mitochondrial benzodiazepine receptors. Endocrinology, in press

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 18:18 For personal use only. No other uses without permission. . All rights reserved.

Regulation of diazepam binding inhibitor in rat adrenal gland by adrenocorticotropin.

Diazepam binding inhibitor (DBI) is a 9-kDa polypeptide that was initially isolated from rat brain and subsequently found to be present in several per...
826KB Sizes 0 Downloads 0 Views