In vivo studies on the role of the peripheral benzodiazepine receptor (PBR) in steroidogenesis

In various steroidogenic cell models, mitochondrial preparations and submitochondrial fractions, the expression of the mitochondrial 18 kDa peripheral-type benzodiazepine receptor (PBR) protein confers the ability to take up and release, upon ligand activation, cholesterol. Thus, cholesterol becomes available to P450scc on the inner mitochondrial membrane. These in vitro studies were validated by in vivo experiments. Treatment of rats with ginkgolide B (GKB), specifically reduced the ligand binding capacity, protein, and mRNA expression of the adrenocortical PBR and circulating glucocorticoid levels. Treatment with GKB also resulted in inhibition of PBR protein synthesis and corticosterone production by isolated adrenocortical cells in response to ACTH. The ontogeny of both PBR binding capacity and protein directly paralleled that of ACTH-inducible steroidogenesis in rat adrenal cells and in rats injected with ACTH. In addition, the previously described suppression of luteal progesterone synthesis in the pregnant rat by continuous in vivo administration of a gonadotropin-releasing hormone agonist may be due to decreased luteal PBR ligand binding and mRNA. These results suggest that (i) PBR is an absolute prerequisite for adrenocortical and luteal steroidogenesis, (ii) regulation of adrenal PBR expression may be used as a tool to control circulating glucocorticoid levels and (iii) the stress hypo-responsive period of neonatal rats may result from decreased adrenal cortical PBR expression.     

Ex vivo regulation of adrenal cortical cell steroid and protein synthesis, in response to adrenocorticotropic hormone stimulation, by the Ginkgo biloba extract EGb 761 and isolated ginkgolide B

We previously demonstrated that repeated treatment of rats with the standardized extract of Ginkgo biloba leaves, EGb 761, and its bioactive component ginkgolide B (GKB), specifically reduces the ligand binding, and protein and messenger RNA expression of the adrenal mitochondrial peripheral benzodiazepine receptor (PBR), a key element in the regulation of cholesterol transport, resulting in decreased circulating corticosterone levels. Adrenocortical cells were isolated from rats treated with EGb 761 or GKB and cultured for 2 and 12 days. The effect of ACTH on normal and metabolically labeled cells was examined. Corticosterone levels were measured by RIA, and protein synthesis was analyzed by two-dimensional gel electrophoresis. Ex vivo treatment with EGb 761 and GKB resulted, respectively, in 50% and 80% reductions of ACTH-stimulated corticosterone production by adrenocortical cells cultured for 2 days compared with that by cells isolated from saline-treated rats. Two-dimensional gel electrophoresis analysis revealed that in cells from both control and drug-treated animals, ACTH induced the synthesis, at the same level, of a 29-kDa and pI 6.4-6.7 protein identified as the adrenal steroidogenic acute regulatory protein (StAR). In addition, treatment with EGb 761 and GKB specifically altered the synthesis of seven proteins, including inhibition of synthesis of a 17-kDa, identified as PBR. After 12 days in culture, ACTH-stimulated adrenocortical cell steroid synthesis was maintained, and it was identical among the cells isolated from animals treated with GKB or saline. Under the same conditions, the expression of PBR was recovered, whereas no effect of ACTH on the 29-kDa and 6.4-6.7 pI protein (StAR) or other protein synthesis could be seen. A comparative analysis of the effects of GKB and EGb 761 on adrenocortical steroidogenesis and protein synthesis identified, in addition to the 17-kDa PBR, target proteins of 32 kDa (pI 6.7) and 40 kDa (pI 5.7-6.0) as potential mediators of the effect of EGb 761 and GKB on ACTH-stimulated glucocorticoid synthesis. In conclusion, these results 1) validate and extend our previous in vivo findings on the effect of EGb 761 and GKB on ACTH-stimulated adrenocortical steroidogenesis, 2) demonstrate the specificity and reversibility of EGb 761 and GKB treatment, 3) question the role of the 29-kDa, 6.4-6.7 pI protein (mature StAR) as the sole mediator of ACTH-stimulated steroid production, and 4) demonstrate the obligatory role of PBR in hormone-regulated steroidogenesis.     

Targeted disruption of the peripheral-type benzodiazepine receptor gene inhibits steroidogenesis in the R2C Leydig tumor cell line

To evaluate the role of the mitochondrial peripheral-type benzodiazepine receptor (PBR) in steroidogenesis, we developed a molecular approach based on the disruption of the PBR gene, by homologous recombination, in the constitutive steroid producing R2C rat Leydig tumor cell line. Inactivation of one allele of the PBR gene resulted in the suppression of PBR mRNA and ligand binding expression. Immunoblot and electron microscopic immunogold labeling analyses confirmed the absence of the 18-kDa PBR protein in the selected clone. Although mitochondria from the PBR-negative cells contained high levels of the constitutively expressed 30-kDa steroidogenic activity regulator protein, these cells produced minimal amounts of steroids compared with normal cells (5%). Moreover, mitochondria from PBR-negative cells failed to produce pregnenolone when supplied with exogenous cholesterol. Addition of the hydrosoluble cholesterol derivative, 22R-hydroxycholesterol, increased steroid production by the PBR-negative R2C cells, indicating that the cholesterol transport mechanism was impaired. Stable transfection of the PBR-negative R2C Leydig cells with a vector containing the PBR cDNA resulted in the recovery of the steroidogenic function of the cells. These data demonstrate that PBR is an indispensable element of the steroidogenic machinery, where it mediates the delivery of the substrate cholesterol to the inner mitochondrial side chain cleavage cytochrome P-450.     

The acute and chronic effects of adrenocorticotropin on the levels of messenger ribonucleic acid and protein of steroidogenic enzymes in rat adrenal in vivo

The purpose of this study was to evaluate the effects of acute (a single injection) and chronic stimulation (twice daily injection for 9 days) by ACTH on changes occurring in the temporal expression of steroidogenic enzymes in the rat adrenal in vivo. Under acute ACTH stimulation, the level of steroidogenic acute regulatory protein (StAR) messenger RNA (mRNA) was increased within 0.5 h in both zona glomerulosa (ZG) and zona fasciculata-reticularis (ZFR), with maximal increases of 220-370% and 300-350% in the ZG and ZFR, respectively. Increases in the levels of StAR protein in homogenates were also found in the ZG (700%) and the ZFR (300%), but were delayed compared with those of their mRNA. Furthermore, the increase in mitochondrial StAR protein was concomitant with that in the homogenate, indicating that the entry of StAR into mitochondria might not be necessary to increase steroidogenesis during the early stimulatory phase. The levels of c-jun, c-fos, junB, and fosB mRNA in ZG and ZFR were also rapidly maximally elevated within 0.5-1 h after ACTH administration and fell to near control levels 5 h posttreatment. The levels of c-jun protein were already increased in both zones at 1 h, reached 200% at 3 h, and remained elevated 5 h post-ACTH treatment. The levels of c-Fos protein were maximally increased by 240% in both zones after 1 h and decreased thereafter to control values at 5 h. Few changes were observed in the adrenal protein contents of cholesterol side-chain cleavage cytochrome P450 (P450scc), cytochrome P450 11beta-hydroxylase (P450C11), cytochrome P450 21-hydroxylase (P450C21), and 3beta-hydroxysteroid dehydrogenase (3betaHSD). Under chronic stimulation by ACTH, we observed elevations in the levels of plasma corticosteroids and changes in the mRNA and protein levels of many adrenal steroidogenic enzymes in both zones. In the ZG, administration of ACTH for 9 days provoked an increase in the level of StAR mRNA (210-270%) and a decrease in the levels of 3betaHSD, cytochrome P450 aldosterone synthase (P450aldo), and AT1 receptor mRNA (by 40%, 70%, and 90%, respectively), whereas the levels of P450scc and P450C21 mRNA did not differ significantly from the control values. Western blotting analysis showed that the adrenal ZG protein levels of StAR and P450scc were increased (150%), 3betaHSD was not changed, and P450C21 was decreased by 70%. In the ZFR, the levels of P450scc and StAR mRNAs were increased (260% and 570-870%, respectively). The levels of 3betaHSD, P450C21, and P450C11 mRNA did not differ from control values in that zone. Western blotting analysis showed that the ZFR protein level of 3betaHSD was not changed, P450scc and P450C21 were decreased by 40% and 60%, respectively, and StAR was increased by 160%. Although c-fos and fosB mRNAs were undetectable after 9 days of chronic ACTH treatment, c-jun mRNA and its protein were still detectable, suggesting a basic role for this protooncogene in maintaining the integrity and function of the adrenal cortex. When dexamethasone was administered to rats for 5 days to inhibit their ACTH secretion, the mRNA levels of many steroidogenic enzymes were decreased, with the exception of StAR, 3betaHSD, and P450aldo. These results confirm the importance of physiological concentrations of ACTH in maintaining normal levels of adrenocortical enzymes and also indicate that in addition to ACTH, other factors are involved in controlling the expression of StAR, 3betaHSD, and P450aldo. In conclusion, we showed that ACTH acutely increases StAR mRNA followed, after a delay, by an increase in the level of StAR protein; this suggests that posttranslational modifications of the StAR precursor occurred during the early stimulatory phase and before the apparent translation of the newly formed mRNA. The rapid induction of protooncogenes suggests their participation in the action of ACTH to stimulate steroidogenesis. (ABSTRACT TRUNCATED)     

Parathyroid hormone (PTH) stimulates adrenocorticotropin release in AtT-20 cells stably expressing a common receptor for PTH and PTH-related peptide

Complementary DNA encoding a rat bone PTH/PTHrP receptor was stably expressed in the murine corticotroph cell line, AtT-20. Several clones, expressing variable numbers of PTH/PTHrP receptors, were developed. In contrast to the relatively low binding affinity (apparent Kd = 15 nM) observed in COS-7 cells transiently expressing the PTH/PTHrP receptor, all AtT-20 stable transfectants bound [Nle8,18,Tyr34]bPTH(1-34)NH2 (NlePTH) with an affinity that was indistinguishable from that observed in ROS 17/2.8 cells expressing native PTH/PTHrP receptors. Additionally, NlePTH dramatically increased cAMP accumulation and ACTH release in AtT-20 cells expressing the PTH/PTHrP receptor with an ED50 of 0.6 +/- 0.3 and 0.3 +/- 0.1 nM, respectively. The high binding affinity and the high efficacy of NlePTH in stimulating cAMP accumulation and ACTH release indicate that the PTH/PTHrP receptor is efficiently coupled to the intracellular signalling system responsible for stimulation of ACTH release in AtT-20 cells. No additivity of cAMP accumulation or of ACTH release was observed when these cells were treated with maximally active concentrations of both NlePTH and CRF. This suggests that the receptors for both of these hormones share the same intracellular effectors, and that intracellular signaling in AtT-20 cells is not compartmentalized. Additionally, the ability of NlePTH to stimulate ACTH release in AtT-20 cells, a function that is normally performed by CRF, demonstrates promiscuity between activated receptors and distal biological functions.     

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Peripheral-type benzodiazepine (BZ) receptors (PBRs) have been identified in various peripheral tissues as well as in glial cells in in the brain. PBRs are located mainly on the outer mitochondrial membrane and bind with high affinity the BZ Ro 5-4864 (4'-cholorodiazepam) and the non-BZ PK 11195 (an isoquinoline carboxamide derivative), but bind with very low affinity the BZ clonazepam. PBRs have been cloned from various species. PBRs are multimeric receptors composed of the 18-kDa binding site for isoquinolines, the 32-kDa voltage-dependent anion channel, and the 30-kDa adenine nucleotide carrier (which binds BZs). The expression of PBRs is especially high in steroidogenic organs. Steroid administration affects PBR density, whereas depletion of hormones by hypophysectomy in female rats, or castration (surgical or chemical) in male rats, decreases PBR density in endocrine organs, which can be elevated to normal values after administration of the appropriate hormone. PBRs are probably involved in several functions, including cell proliferation, respiration, and steroidogenesis. It has been suggested that PBRs are involved in the translocation of cholesterol from the outer to the inner membrane of the mitochondria and have an effect on the biosynthesis of steroids.     

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Steroidogenic acute regulatory protein (StAR), a 30-kDa protein involved in the transport of cholesterol to inner mitochondrial membrane during stimulation of steroid hormone biosynthesis, has recently been cloned from human adrenals and MA-10 mouse Leydig tumor cells. We examined the regulation of StAR mRNA accumulation upon induction of steroidogenesis in immortalized rat granulosa cells. Granulosa cells were transfected with SV40 DNA alone (POGS5); with SV40 DNA and Ha-ras oncogene (POGRS1); with SV40 DNA, Ha-ras oncogene and LH/CG receptor (GLHR15) or with FSH receptor (GFSHR17) or with the beta 2-adrenergic receptor (G beta 2AR13) expression plasmids. Cells were cultured to confluency and then stimulated for 24 h with oFSH (4 nM), hCG (2.4 nM), isoproterenol (10 microM) or forskolin (50 microM). By quantitative RT-PCR, StAR mRNA was undetectable in non-steroidogenic cells (transfected with SV40 DNA alone, POGS5) either in the presence or in the absence of forskolin. In contrast, variable amount of the message was detected in all steroidogenic cell lines cotransfected with SV40 DNA and Ha-ras. Moreover, an increase in the StAR mRNA expression was evident in all steroidogenic cells upon stimulation with their respective agonists, concomitantly with enhanced progesterone production. The RT-PCR product was sequenced and the 379 base pairs of rat StAR were found to be 93% and 86% identical to mouse and human cDNA, respectively. The deduced 126 amino acid sequence was 95%, 88% and 88% identical to the mouse, human and bovine deduced protein sequences. We conclude that StAR message is expressed only in the steroidogenic rat granulosa cells and can be upregulated by FSH, hCG, isoproterenol and forskolin in the appropriate cell lines. In addition, we find that the rat StAR cDNA exhibit a high degree of homology with the mouse and human sequences.     

Peripheral-type benzodiazepine receptor function in cholesterol transport. Identification of a putative cholesterol recognition/interaction amino acid sequence and consensus pattern

In steroid-synthesizing cells, like the MA-10 mouse tumor Leydig cells, the peripheral-type benzodiazepine receptor (PBR) is an outer mitochondrial membrane protein involved in the regulation of cholesterol transport from the outer to the inner mitochondrial membrane, the rate-determining step in steroid biosynthesis. Expression of PBR in Escherichia coli DE3 cells, which have no PBR, no cholesterol, and do not make steroids, induced the ability to take up cholesterol in a time-dependent, temperature-sensitive, and energy-independent manner. These cells took up no other steroids tested. Addition of the high affinity PBR ligand PK 11195 to cholesterol-loaded membranes, obtained from cells transfected with PBR, resulted in the release of the uptaken cholesterol. Expression in DE3 cells of mutant PBRs demonstrated that deletions in the cytoplasmic carboxy-terminus dramatically reduced the cholesterol uptake function of PBR, although it retained full capacity to bind PK 11195. Site-directed mutagenesis in the carboxy-terminal region of PBR demonstrated that bacteria expressing the mutant PBR proteins PBR(Y153S) and PBR(R156L) do not accumulate cholesterol, suggesting that amino acids Y153 and R156 are involved in the interaction of the receptor with cholesterol. Considering these results, we postulate the existence of a common cholesterol recognition/interaction amino acid consensus pattern (-L/V-(X)(1-5)-Y-(X)(1-5)-R/K-). Indeed, we found this amino acid consensus pattern in all proteins shown to interact with cholesterol. In conclusion, these data suggest that the expression of PBR confers the ability to take up and release, upon ligand activation, cholesterol. Considering the widespread occurrence of this protein and its tissue and cell specific subcellular localization, these results suggest a more general role of PBR in intracellular cholesterol transport and compartmentalization.     

Acute action of choriogonadotropin on Leydig tumor cells: changes in the topography of the mitochondrial peripheral-type benzodiazepine receptor

We examined the topography of the MA-10 Leydig tumor cell mitochondrial peripheral-type benzodiazepine receptor (PBR). In previous studies, the 18 kDa PBR was found to be functionally associated with the voltage-dependent anion channel, located in the junctions between outer and inner membranes. Transmission electron (TEM) and atomic force microscopy (AFM) of immunogold labeled PBR on Leydig cell mitochondrial preparations showed that the 18 kDa PBR protein is organized in clusters of 4-6 molecules. Addition of hCG to Leydig cells induces a rapid, within 30 sec, increase in PBR ligand binding and morphological changes, namely redistribution of PBR molecules in large clusters (>7 particles). These hCG-induced changes were inhibited by a cAMP-dependent protein kinase inhibitor and by the benzodiazepine flunitrazepam. AFM further demonstrated the rapid reorganization of the mitochondrial membrane, where the formation of contacts between the outer and the inner mitochondrial membrane may facilitate cholesterol transfer.     

Interferon-gamma inhibits the steroidogenic acute regulatory protein messenger ribonucleic acid expression and protein levels in primary cultures of rat Leydig cells

Interferon-gamma (IFNgamma) is an immunomodulating cytokine that has profound effects on reproductive function. IFNgamma inhibits steroidogenesis both in vivo and in vitro. The mechanism by which IFNgamma inhibits Leydig cell steroidogenesis remains unclear. In the present study, we evaluated the effect of IFNgamma on the expression and regulation of the steroidogenic acute regulatory protein (StAR) gene in primary cultures of rat Leydig cells. StAR facilitates the efficient production of steroid hormone by regulating the translocation of cholesterol from the outer to the inner mitochondrial membrane, the site of the cytochrome P450 side-chain cleavage (P450scc) enzyme system that converts cholesterol to pregnenolone. IFNgamma inhibited hCG-induced StAR messenger RNA (mRNA) levels in a dose-dependent manner. The addition of IFNgamma in a concentration of 500 U/ml decreased hCG-induced 3.8- and 1.7-kilobase StAR mRNA by 78% and 70%, respectively. IFNgamma also reduced hCG-stimulated P450scc mRNA levels by 69%. The inhibitory effects of IFNgamma on StAR mRNA levels were confirmed by ribonuclease protection assay. As early as 12 h after the addition of IFNgamma, hCG-induced StAR mRNA levels decreased by more than 44%. To evaluate the effects of IFNgamma on StAR protein levels, Western blot analyses were performed. hCG in a concentration of 10 ng/ml increased StAR protein by 5.6-fold. Treatment of Leydig cells with IFNgamma (500 U/ml) decreased hCG-induced StAR protein by 44%. In contrast, interleukin-1 and murine tumor necrosis factor-alpha reduced hCG-induced P450scc mRNA expression without inhibiting StAR mRNA or protein levels. In conclusion, IFNgamma inhibits Leydig cell steroidogenesis by down-regulating StAR gene expression and protein production.     

Submitochondrial distribution of three key steroidogenic proteins (steroidogenic acute regulatory protein and cytochrome p450scc and 3beta-hydroxysteroid dehydrogenase isomerase enzymes) upon stimulation by intracellular calcium in adrenal glomerulosa cells

In adrenal glomerulosa cells, angiotensin II (Ang II) and potassium stimulate aldosterone synthesis through activation of the calcium messenger system. The rate-limiting step in steroidogenesis is the transfer of cholesterol to the inner mitochondrial membrane. This transfer is believed to depend upon the presence of the steroidogenic acute regulatory (StAR) protein. The aim of this study was 1) to examine the effect of changes in cytosolic free calcium concentration and of Ang II on intramitochondrial cholesterol and 2) to study the distribution of StAR protein in submitochondrial fractions during activation by Ca2+ and Ang II. To this end, freshly prepared bovine zona glomerulosa cells were submitted to a high cytosolic Ca2+ clamp (600 nM) or stimulated with Ang II (10 nM) for 2 h. Mitochondria were isolated and subfractionated into outer membranes, inner membranes (IM), and contact sites (CS). Stimulation of intact cells with Ca2+ or Ang II led to a marked, cycloheximide-sensitive increase in cholesterol in CS (to 143 +/- 3. 2 and 151.1 +/- 18.1% of controls, respectively) and in IM (to 119 +/- 5.1 and 124.5 +/- 6.5% of controls, respectively). Western blot analysis revealed a cycloheximide-sensitive increase in StAR protein in mitochondrial extracts of Ca2+-clamped glomerulosa cells (to 159 +/- 23% of controls). In submitochondrial fractions, there was a selective accumulation of StAR protein in IM following stimulation with Ca2+ (228 +/- 50%). Similarly, Ang II increased StAR protein in IM, and this effect was prevented by cycloheximide. In contrast, neither Ca2+ nor Ang II had any effect on the submitochondrial distribution of cytochrome P450scc and 3beta-hydroxysteroid dehydrogenase isomerase. The intramitochondrial presence of the latter enzyme was further confirmed by immunogold staining in rat adrenal fasciculata cells and by immunoblot analysis in MA-10 mouse testicular Leydig cells. These findings demonstrate that under acute stimulation with Ca2+-mobilizing agents, newly synthesized StAR protein accumulates in IM after transiting through CS. Moreover, our results suggest that the import of StAR protein into IM may be associated with cholesterol transfer, thus promoting precursor supply to the two first enzymes of the steroidogenic cascade within the mitochondria and thereby activating mineralocorticoid synthesis.     

Characterization of an epithelial approximately 460-kDa protein that facilitates endocytosis of intrinsic factor-vitamin B12 and binds receptor-associated protein

By using receptor-associated protein (RAP) as an affinity target, an intrinsic factor-vitamin B12 (IF-B12)-binding renal epithelial protein of approximately 460 kDa was copurified together with the transcobalamin-B12-binding 600-kDa receptor, megalin. IF-B12 affinity chromatography of renal cortex membrane from rabbit and man yielded the same approximately 460-kDa protein. Binding studies including surface plasmon resonance analyses of the protein demonstrated a calcium-dependent and high affinity binding of IF-B12 to a site distinct from the RAP binding site. The high affinity binding of IF-B12 was dependent on complex formation with vitamin B12. Light and electron microscope autoradiography of rat renal cortex cryosections incubated directly with IF-57Co-B12 and rat proximal tubules microinjected in vivo with the radioligand demonstrated binding of the ligand to endocytic invaginations of proximal tubule membranes followed by endocytosis and targeting of vitamin B12 to lysosomes. Polyclonal antibodies recognizing the approximately 460-kDa receptor inhibited the uptake. Immunohistochemistry of kidney and intestine showed colocalization of the IF-B12 receptor and megalin in both tissues. In conclusion, we have identified the epithelial IF-B12-binding receptor as a approximately 460-kDa RAP-binding protein facilitating endocytosis.     

Environmentally induced changes in peripheral benzodiazepine receptors are stressor and tissue specific

The stress-induced changes in peripheral benzodiazepine receptors (PBR) can be observed in a number of different tissues, depending upon the nature and chronicity of the aversive experience. In addition, virtually all stress procedures that cause rapid changes in PBR simultaneously increase the physical activity or metabolic rate of the subjects. The present study analyzed the contributions of rapid alterations in activity or metabolic rate with and without aversive stimulation and their subsequent impact on PBR. Mechanically induced increases in activity by forced running stress results in a significant reduction in [3H]Ro 5-4864 binding to PBR in olfactory bulb, opposite to the PBR changes in this tissue following forced cold-water swim stress. Pharmacological induction of increased locomotor activity as well as metabolic rate by d-amphetamine causes a significant increase in cardiac PBR binding, again, opposite to the response typically observed following inescapable shock stress. Finally, administration of the anxiogenic beta-carboline, FG-7142, causes increases in both hippocampus and adrenal gland PBR binding reminiscent of acute noise stress exposure. These experiments demonstrate that increased locomotor activity or metabolic rate alone is not a necessary and sufficient condition for previous stress-induced changes in PBR. Conversely, increased metabolic rate coupled with an aversive stimulus appears to be an important factor for inducing stress-like changes in PBR. This data, coupled with previous reports, suggests that rapid alterations in these sites are stressor and tissue dependent. Finally, we propose that the PBR may be involved in many aspects of the stress response including: a) a blowarning system in adrenal gland, b) participation in stress-induced hypertension via renal PBR, and c) a modulator of stress-induced immunosuppression and subsequent recovery of function or recuperation by actions on immune cells.     

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The displacement of porcine [125I] insulin bound to rat and lamprey isolated hepatocytes with unlabeled lamprey and porcine insulins was investigated. Binding affinity of lamprey insulin for insulin receptor of rat was similar to that of porcine insulin. In contrast, the binding affinity of lamprey insulin for its own insulin receptor was higher than for a rat receptor. To determine the binding affinity constants of lamprey insulin receptor, the competition binding experiments were carried out on isolated lamprey hepatocytes using lamprey insulin as unlabeled ligand and tracer. The affinity of the same binding sites on lamprey hepatocytes was assessed in similar experiments but employing porcine insulin as unlabeled ligand and tracer. It was found that while Kd of low affinity binding sites on lamprey hepatocytes were similar for lamprey and porcine insulins, the Kd of high affinity binding sites was different: the displacement curve for lamprey insulin being shifted to the left as compared to the curve for porcine insulin. The number of high and low affinity binding sites, calculated independently in Scatchard plots, was equal. We conclude that the high affinity insulin binding sites of lamprey but not of rat hepatocytes reveal some species specificity in ligand-receptor interaction.     

Receptors for calcitonin gene-related peptide (CGRP) in the rat adrenal cortex

We previously demonstrated the presence of adrenomedullin receptors in the rat adrenal cortex. There is evidence, however, that the actions of adrenomedullin may also be mediated by the CGRP receptor. The present study was designed to determine whether specific CGRP receptors are present in the rat adrenal cortex. Adrenal glands were, sectioned and immunostained with a primary antibody raised against the first intracellular loop of the CGRP-I receptor. Staining was visualised using alkaline phosphatase and vector red. Immunostaining for the CGRP-I receptor was found in the zona glomerulosa and the adrenal medulla, but not in the inner adrenocortical zones. ACTH treatment caused an increase in staining intensity in the glomerulosa. Ligand binding studies suggested the existence of two populations of CGRP binding sites, one with a Kd of 0.1 nM, the second of 37 nM. Only CGRP-I and adrenomedullin displaced labeled CGRP binding. These results suggest that the CGRP-I receptor is expressed in the adrenal zona glomerulosa and that a second class of binding site is also present. The CGRP-I receptor appears to be regulated by ACTH.     

Ethane dimethane sulfonate and NNN'N'-tetrakis-(2-pyridylmethyl)ethylenediamine inhibit steroidogenic acute regulatory (StAR) protein expression in MA-10 Leydig cells and rat Sertoli cells

Apoptosis inhibits steroid biosynthesis, but it is not clear how the Steroidogenic Acute Regulatory (StAR) protein, is affected. To characterize StAR expression during apoptosis, mouse MA-10 Leydig tumor cells were treated with ethane dimethane sulfonate (EDS), an inducer of apoptosis, and the metal ion chelator NNN'N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), an inducer of cell death. Both chemicals induced cell death and similarly inhibited dbcAMP-stimulated steroidogenesis and accumulation of the 30 kDa form of StAR. Utilizing the dye JC-1, it was found that TPEN and EDS also impaired the mitochondrial electrochemical potential (delta psi). In Sertoli cells, which also express StAR, EDS induced cell death and attenuated StAR expression. We conclude 1) steroidogenesis and accumulation of mature StAR protein are inhibited as a consequence of the induction of apoptosis; 2) reduced levels of StAR may be partially attributed to inhibition of import because of the loss of delta psi; 3) loss of steroidogenesis is probably due to loss of StAR synthesis and disruption of delta psi.     

Functioning of the porcine pituitary-adrenocortical axis during neonatal development

A study was conducted with neonatal boars to measure age-related changes in functioning of the pituitary-adrenocortical axis. Pigs were randomly assigned to control (n = 7-10/age) or treated (1-min restraint, n = 9-11/age) groups to be sampled at either 12, 19, or 26 days of age. Blood samples were taken via catheter 10 min before and 3, 10, and 20 min after restraint or at similar time intervals in controls. One day later, pigs were killed and adrenal glands obtained for ACTH receptor measurements. Basal plasma ACTH concentrations were greatest (p = 0.035) on day 12 when compared with later ages, but basal plasma cortisol concentrations were comparable at the three ages. Compared with controls, restraint elevated incremental plasma ACTH and cortisol responses at each age (p < 0.004). On day 12, maximal plasma ACTH (p = 0.0006) and incremental cortisol (p < 0.006) responses to restraint were greater than at later ages. Binding to adrenal ACTH receptors was greatest (p < 0.05) at day 13, which may help explain the apparently increased in vivo response of the adrenal gland to ACTH at this time. Restrained pigs had increased growth rates with increasing age (p = 0.016) whereas growth rates for control pigs did not differ with age. At day 27, 24 h after the 1-min restraint, body weights of restrained pigs exceeded those of control pigs (p = 0.045). At day 20, adrenal DNA and protein in pigs restrained 24 h previously were greater than in control pigs (p < 0.05). These data suggest age-related changes in functioning of the pituitary-adrenal axis in neonatal boars, and an absence of period during neonatal life when the porcine pituitary adrenocortical axis cannot respond to a stressor. The data also indicate both rapid and long-term responses of the adrenal to a very modest stressor and suggest an extreme sensitivity of neonatal pigs to environmental perturbations.     

ATP and a mitochondrial electrochemical gradient are required for functional activity of the steroidogenic acute regulatory (StAR) protein in isolated mitochondria

The Steroidogenic Acute Regulatory (StAR) protein has been put forth as the rapidly synthesized, cycloheximide-sensitive protein that is required for the transport of cholesterol to the inner mitochondrial membrane and the P450scc enzyme and thereby acutely regulates steroidogenesis in steroidogenic tissues. In this study, several of the factors that may be required for StAR activity were examined using an in vitro system. Lysates from StAR-transfected COS-1 cells were added to mitochondria isolated from MA-10 Leydig tumor cells. Results obtained demonstrated that StAR-containing cell lysate increased steroidogenesis in isolated mitochondria, but failed to do so in the presence of m-CCCP, apyrase, or AMP-PNP, suggesting that StAR function requires ATP hydrolysis as well as an electrochemical gradient for maximal steroidogenic activity.     

Steroidogenic acute regulatory protein (StAR) acts on the outside of mitochondria to stimulate steroidogenesis

Steroidogenic acute regulatory protein (StAR) facilitates delivery of cholesterol to the inner mitochondrial membranes. StAR is imported into mitochondria and processed to a mature form by cleavage of the N-terminal mitochondrial targeting sequence. We produced His-tagged (His-tag StAR) constructs lacking the N-terminal 62 amino acids that encode the mitochondrial targeting sequence and examined their steroidogenic activity in intact cells and on isolated mitochondria. His-tag StAR proteins stimulated pregnenolone synthesis to the same extent as wild-type StAR when expressed in COS-1 cells transfected with the cholesterol side-chain cleavage system. His-tag StAR was diffusely distributed in the cytoplasm of transfected COS-1 cells, whereas wild-type StAR was localized to mitochondria. There was no evidence at the light or electron microscope levels for selective localization of His-tag StAR protein to mitochondrial membranes. We established an assay system using mitochondria isolated from bovine corpora lutea and purified recombinant His-tag StAR proteins expressed in E. coli. Recombinant His-tag StAR stimulated pregnenolone production in a dose- and time-dependent manner, functioning at nanomolar concentrations. A point mutant of StAR (A218V) that causes lipoid congenital adrenal hyperplasia was incorporated into the His-tag protein. This mutant was steroidogenically inactive in COS-1 cells and on isolated mitochondria. Our observations conclusively document that StAR acts on the outside of mitochondria, independent of mitochondrial import.