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.     

Structure and function of the peripheral-type benzodiazepine receptor in steroidogenic cells

Steroidogenesis depends on the rate of cholesterol transport from intracellular stores to the inner mitochondrial membrane cytochrome P-450 side-chain cleavage enzyme. Using steroidogenic cell submitochondrial fractions, mitochondrial preparations, various cell models, and animal models and with the help of pharmacological, biochemical, morphological, and molecular approaches, we provide evidence that the peripheral-type benzodiazepine receptor mediates the intramitochondrial cholesterol transport and the subsequent adrenal, gonadal, placental, and brain steroid biosynthesis.     

Identification and characterization of a high-affinity peripheral-type benzodiazepine receptor in rabbit urinary bladder

The present study used radioligand binding and in vitro contractility experiments to identify and characterize a peripheral-type benzodiazepine receptor PBR in rabbit urinary bladder. [3H]PK11195 bound to bladder membranes with high-affinity and density (Kd = 5.2 nM., Bmax = 268 fmol./mg. protein), indicating the presence of a PBR. [3H]flunitrazepam bound with high-affinity and density (Kd = 1.2 nM., Bmax = 48 fmol./mg. protein). The rank order potency of various benzodiazepines and isoquinoline carboxamides in displacing the binding of [3H]PK11195 was Ro5-4864 > diazepam = flunitrazepam > Ro15-1788 = clonazepam. Ro5-4864 and PK11195 inhibited nerve-evoked contractions in a concentration-dependent manner (IC50 = 42 microM. and 56 microM., respectively). Carbachol- and KCl-induced contractions were also inhibited by Ro5-4864 and PK11195. KCl-induced contractions were inhibited to a greater extent than carbachol-induced or field-stimulated contractions with all the drugs tested. Both Ro5-4864 and PK11195 significantly increased the ED50 for calcium-induced contractions following a cholinergic stimulus compared with control. These data demonstrate the presence of a PBR in urinary bladder capable of altering contractility in vitro through modulation of calcium activity.     

Involvement of peripheral-type benzodiazepine receptors in the intracellular transport of heme and porphyrins

To investigate the involvement of peripheral-type benzodiazepine receptors (PBR) in heme metabolism, we examined the interaction of [55Fe]heme with PBR. Transfection of the cloned mouse PBR-isoquinoline carboxamide-binding protein (PBR/IBP) cDNA into monkey kidney Cos-1 cells resulted in a 2.5-fold increase in [55Fe]hemin binding sites, concomitant with the increase in [3H]PK11195 binding sites, as compared with those seen in antisense PBR/IBP cDNA-transfected cells. The binding of hemin to the transfected receptors exhibited a relatively high affinity with a Kd of 12 nM, and was inhibited by several benzodiazepine ligands, including PK11195, Ro 5-4864, diazepam and protoporphyrin IX. When mouse liver mitochondria were incubated with [55Fe]hemin, the binding to PBR had a Kd of 15 +/- 1.8 nM. The Bmax of [55Fe]hemin binding to the mitochondria was 6.88 +/- 0.76 pmol/mg of protein, a value consistent with that of [3H]PK11195 binding, with a lower affinity. Coproporphyrinogen III, a precursor porphyrin produced in the cytosol, is translocated into mitochondria, then is converted to protoporphyrinogen IX; this conversion decreased in the presence of benzodiazepine ligands. To examine whether this decrease was related to a decrease in the binding of coproporphyrinogen to the mitochondria, the effects of benzodiazepines on the binding of coproporphyrinogen were examined. As the binding was dose-dependently inhibited by PK11195, Ro 5-4864, and diazepam, porphyrins are likely to be endogenous ligands for PBR. We propose that PBR play a role in the intracellular transport of porphyrins and heme.     

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.     

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.     

Decreases in peripheral-type benzodiazepine receptors in postmortem brains of chronic schizophrenics

We measured the peripheral-type benzodiazepine receptors (PBRs), a marker of gliosis, in 26 brain areas (cerebral cortex, thalamus and extrapyramidal system) of the postmortem brains of 13 chronic schizophrenics and 10 controls, using [3H] PK 11195 as a ligand for the receptor assay. The specific [3H] PK 11195 binding was significantly decreased in three brain areas (superior parietal cortex, primary visual area and putamen) of schizophrenics, although there were no changes in the binding in the other brain areas. Scatchard analysis revealed that there were decreases in both the Bmax and Kd of [3H] PK 11195 binding in the brain areas. These results were almost in accordance with a number of neuropathological studies reporting that there was no change or reduction in glial cells in the brain regions of schizophrenics and suggested that the decreased density of PBRs in the brain may be involved in the pathophysiology of schizophrenia, associated with reduced production of neurosteroids coupled to PBRs.     

Developmental expression of alpha 9 acetylcholine receptor mRNA in the rat cochlea and vestibular inner ear

Expression of alpha9 acetylcholine receptor (AChR) mRNA was studied by in situ hybridization in the rat adult and developing cochlea and vestibular inner ear. Alpha9 AChR mRNA was first observed in cochlear hair cells (HCs) at embryonic day 18 (E18), increased markedly after birth, stayed high until postnatal day 10 (P10), and decreased to substantially lower adult levels by P14. High levels of alpha9 AChR mRNA expression were also noted in the developing nonneuronal structures of the inner sulcus, chondrocytes, and/or osteoblasts in the cochlear capsule and interscalar laminae. Both developing and adult bone marrow cells also expressed intense alpha9 AChR mRNA. In the vestibular system, alpha9 AChR mRNA was first observed in HCs at E16 in all sensory epithelia, increased to its highest levels by P0-P4, then decreased slightly to reach adult levels by P10. The results are consistent with the alpha9 AChR subserving efferent neurotransmission to both cochlear and vestibular HCs. The observation of alpha9 AChR mRNA in cochlear HCs 2 weeks prior to functional onset in the cochlea further suggests that expression of this gene is not related to HC activity. The observation of substantial nonneuronal expression of alpha9 AChR mRNA suggests that this receptor also has functions separate from its role in neurotransmission.     

Induction of peripheral-type benzodiazepine receptors in mouse brain following thioacetamide-induced acute liver failure

To investigate the possible role of peripheral-type benzodiazepine receptors (PBR) in hepatic encephalopathy, we examined expression of PBR in mouse brain following thioacetamide (TAA)-induced acute liver failure. Treatment of mice with TAA resulted in an increase in the number of binding sites of the PBR ligand [3H] Ro5-4864 to brain homogenates, with no significant change in affinity of the ligand. The order of potency of different ligands to compete against [3H] Ro5-4864 binding in the brain of TAA-treated mice was Ro5-4864 > PK11195 > diazepam > protoporphyrin IX, findings similar to those in the control. Northern blot analysis revealed an increase in PBR/isoquinoline binding protein (PBR/IBP) mRNA in mouse brain following TAA treatment, in a time- and dose-dependent manner. These results indicate that the increased number of PBR in the brains of TAA-treated mice relates to the induction of PBR/IBP expression and suggest that the induction of PBR in brain may contribute to pathogenesis of hepatic encephalopathy.     

Changes in the adrenal glands in menigococcemia

Post-mortem morphological, histochemical, and morphometric investigations of the adrenals in 46 deceased patients who suffered from meningococcemia with and without lesions of the meninges were carried out. The control group was composed of 7 practically healthy persons who had perished suddenly as a result of craniocerebral trauma. It was shown that the adrenals might often be seriously damaged in meningococcemia without clinical and morphological signs of meningitis. Hemodynamic disorders, in particular, involvement of the vessels of the microcirculatory bed, contributed considerably to the lesions of the adrenals. However, even in meningococcemia free from signs of meningitis, in a number of cases individual areas of the cortex retained a high functional activity. In meningococcemia combined with lesions of the meninges, despite considerable damage of the cortical layer, in the majority of cases there were evidences of the tense functioning of the adrenal cells.     

Venous infarction of the adrenal glands

An investigation of 78 cases of adrenal haemorrhage and necrosis disclosed that 32 were examples of adrenal venous infarction. In all these cases there was thrombosis of the main adrenal vein and in most there was also thrombosis of the capsular veins, a finding which has not been well established. In a number of cases with venous infarction there was clinical and pathological evidence that disseminated intravascular coagulation (DIC) had occurred, but it appears that it was not the direct cause of venous thrombosis. The majority of cases of venous infarction occur in patients with severe infection, frequently of the respiratory tract. Venous infarction was found in five cases with hypothermia an association which had rarely been described, and in three of these there was evidence of DIC. This is apparently the first occasion on which DIC has been demonstrated in cases of hypothermia in man. The cause of venous thrombosis in the adrenal glands is obscure in most cases of venous infarction, although in three it was due to involvement by metastatic carcinoma. It is suggested that the factors responsible for the initiation of thrombosis in the adrenal veins are catecholamines, thrombin, fibrin and endotoxin. Localisation of the thrombi to the adrenal vein is due to the unique anatomical structure of the vein which, under certain circumstances, results in the local stasis of blood.     

Comparative characteristics of steroidogenesis and its biorhythm in the hyperfunctioning adrenal cortex

The paper summarizes the results od several studies of the daily rhythms of steroid hormones in patients with ACTH-dependent Itsenko-Cushing's disease (CD) and congenital adrenal hyperplasia (late-onset forms) (CAH). Normal daily rhythms of adrenal C21- and C19-steroids and ACTH were observed in 23.5% of CD patients. CAH patients had the marked daily rhythms of adrenal androgens and testosterone which were typical of those of cortisole. The ratios of steroid hormones to its precursors provide evidence for enhanced activities of 17-, 11 beta- and 18-hydroxylases in CD patients and normal enzymatic activities in CAH patients, whereas 21-hydroxylase being an exception.     

Bilateral non-Hodgkin's lymphoma of the adrenal glands with adrenal insufficiency

A 66-year-old female presented with anorexia, fatigue, skin pigmentation, weight loss and low grade fever. Imaging studies demonstrated bilateral bulky masses confined to the adrenal glands. Ultrasonography guided needle biopsy of the mass showed findings of diffuse large B-sell lymphoma. Low levels of serum cortisol, urinary 17-OHCS and 17-KS, a high level of ACTH and a non-reactive pattern on the rapid ACTH test led to a diagnosis of Addison's disease. Only a partial regression was achieved by the first chemotherapy. She died due to disease progression, while the next course of chemotherapy had been postponed because of interstitial pneumonitis due to methotrexate.     

The involvement of innervation in the regulation of fetal adrenal steroidogenesis

Over the last decade, great interest has been generated in evaluation of the extent of neural control of the adrenal cortex and in adrenal cortical/medullary paracrine interactions. The purpose of this review is to summarize current knowledge of fetal adrenal cortical innervation and to present an overview of those studies of fetal adrenal function indicating that adrenal innervation plays a functional role in the control of glucocorticoid secretion under basal conditions and in response to a variety of homeostatic challenges. It will be helpful in understanding both the innervation of the adrenal cortex and the role of adrenal innervation in steroidogenesis during fetal development to briefly review experimental studies that have shed light on adrenal steroidogenesis during postnatal life. This is helpful for two reasons: 1) the vast majority of studies of adrenal innervation and its effect on steroidogenesis have utilized postnatal animals and 2) since the fetus is preparing for postnatal life, evaluating the level of function achieved postnatally provides crucial insights into the developmental stages of adrenal innervation and its role in steroidogenesis in preparing the fetus for an independent postnatal existence.     

Inhibition of morphine-induced tolerance and dependence by a benzodiazepine receptor agonist midazolam in the rat

We investigated whether midazolam administration influenced morphine-induced antinociception and tolerance and dependence in the rat. Antinociception was assessed by the tail-flick (TF) and the hot-plate test (HP 52 degrees C). Morphine tolerance developed after daily single injections of morphine for 11 days. The effect of midazolam on morphine-induced antinociception and tolerance was assessed by giving daily injections of various doses of midazolam for 11 days. The first injection of saline or midazolam was given intraperitoneally and 30 min later morphine (10 mg/kg body weight) was administered subcutaneously. Antinociception was monitored by measuring TF and HP latencies 60 min after the second injection. Midazolam was injected at four different concentrations: 0.03, 0.1, 0.3, and 3 mg/kg body weight. Chronic administration of morphine resulted in the development of tolerance to antinociception in both TF and HP tests, with rats exhibiting baseline antinociception on Day 9. Animals treated with midazolam alone showed little antinociception on Days 3-9. However, midazolam administration in morphine-treated animals attenuated morphine-induced tolerance to antinociception on Days 1-11 as measured by the tail-flick test. Midazolam also decreased the jumping behavior following naloxone injections in morphine-dependent rats. These results suggest that midazolam may prolong the effects of morphine by delaying morphine-induced development of tolerance to antinociception. Midazolam also attenuated a decrease in weight gain induced by chronic injections of morphine.     

Pseudocysts of the adrenal glands in newborn infants

With diagnostic aims the author used direct laryngeal tomography in patients with median stenosis of the larynx. Tomography made it possible to define the width of the lumen of the sumplica area. Depending on the change of the subplica area the author varied the extent of surgical intervention. He used the extralaryngeal method of vocal plica laterification in all patients. With narrow subglottis he practiced complete mobilization of the elastic cone and its subsequent laterification; with wide subglottis he mobilized only the uper edge of the elastic cone. He succeeded in obtaining good functional results concerning respiration in all patients.     

Peripheral benzodiazepine receptor expression on leukocytes and neutrophil function during anticonvulsant monotherapy

AIM: To gain a full understanding of the concept of equity and means of promoting and monitoring a more equitable healthservice. BACKGROUND: With previous policy decisions having sidelined equity, a new government is likely to wish to reverse this trend. ORIGINS OF INFORMATION: Government and expert opinion. DATA ANALYSIS: A critical approach to previous literature. KEY ISSUES: Equity is not a unitary concept and so it is explored and dissected using the framework of: Distribution of Resources, Access and Uptake of Healthcare, Standards and Outcomes of Healthcare, Health Status. Means of improving and monitoring equity in these four areas are suggested. CONCLUSIONS: There are a number of ways of considering the concept of equity and for any fruitful discussion to occur there needs to be an agreed working definition. Specific topics are discussed where equity is lacking and a plan of action is suggested which is likely to improve equity in the areas identified.     

Regulation of immunoreactive androgen receptor in the adrenal gland of the adult rat

The androgen receptor (AR) was measured by an immunoblot assay in adult tissues of both male and female rats. Relatively high levels of AR were detected in tissues of the male urogenital tract and in the adrenal glands and gonads of both sexes. Another group of tissues, including the male levator ani/bulbocavernosus muscles, preputial gland, scrotal skin, and vagina, had low, but detectable, levels of AR. In a third group of tissues, including the uterus, kidney, spleen, liver, gut, heart, lung, pituitary, and hypothalamus, AR was undetectable. In some androgen target tissues, such as the penis, androgens cause an apparent disappearance of AR from the tissue, and in other tissues, such as the ventral prostate, androgen therapy increases the amount of detectable AR. We compared the effect of androgen on AR levels in the adrenal gland and ventral prostate, tissues that differ markedly in their trophic responses to androgen. Castration appeared to have no effect on the amount of detectable AR in the adrenal gland, whereas it caused a profound decrease in AR levels in the ventral prostate. By contrast, 7 days after hypophysectomy, AR levels declined in both the adrenal gland and the ventral prostate. The effects of hypophysectomy plus castration were similar to those of hypophysectomy alone. Administration of ACTH to hypophysectomized rats for 7 days did not reverse the effects of hypophysectomy on adrenal AR, nor did treatment with levothyroxine, dexamethasone, rat GH, or rat PRL. Treatment of hypophysectomized rats with 5alpha-dihydrotestosterone for 7 days caused a dramatic increase in the amount of detectable AR in both the ventral prostate and the adrenal gland, but had a trophic effect only in the ventral prostate. These findings suggest that the amount of immunoreactive AR detected in both the adrenal gland and the ventral prostate is enhanced by androgens: testicular androgens in the case of the ventral prostate and adrenal androgen in the case of the adrenal glands.