Disruption of the glucocorticoid receptor gene in the nervous system results in reduced anxiety.

The glucocorticoid receptor (Gr, encoded by the gene Grl1) controls transcription of target genes both directly by interaction with DNA regulatory elements and indirectly by cross-talk with other transcription factors. In response to various stimuli, including stress, glucocorticoids coordinate metabolic, endocrine, immune and nervous system responses and ensure an adequate profile of transcription. In the brain, Gr has been proposed to modulate emotional behaviour, cognitive functions and addictive states. Previously, these aspects were not studied in the absence of functional Gr because inactivation of Grl1 in mice causes lethality at birth (F.T., C.K. and G.S., unpublished data). Therefore, we generated tissue-specific mutations of this gene using the Cre/loxP -recombination system. This allowed us to generate viable adult mice with loss of Gr function in selected tissues. Loss of Gr function in the nervous system impairs hypothalamus-pituitary-adrenal (HPA)-axis regulation, resulting in increased glucocorticoid (GC) levels that lead to symptoms reminiscent of those observed in Cushing syndrome. Conditional mutagenesis of Gr in the nervous system provides genetic evidence for the importance of Gr signalling in emotional behaviour because mutant animals show an impaired behavioural response to stress and display reduced anxiety.

Mice with an increased glucocorticoid receptor gene dosage show enhanced resistance to stress and endotoxic shock.

Targeted mutagenesis of the glucocorticoid receptor has revealed an essential function for survival and the regulation of multiple physiological processes. To investigate the effects of an increased gene dosage of the receptor, we have generated transgenic mice carrying two additional copies of the glucocorticoid receptor gene by using a yeast artificial chromosome. Interestingly, overexpression of the glucocorticoid receptor alters the basal regulation of the hypothalamo-pituitary-adrenal axis, resulting in reduced expression of corticotropin-releasing hormone and adrenocorticotrope hormone and a fourfold reduction in the level of circulating glucocorticoids. In addition, primary thymocytes obtained from transgenic mice show an enhanced sensitivity to glucocorticoid-induced apoptosis. Finally, analysis of these mice under challenge conditions revealed that expression of the glucocorticoid receptor above wild-type levels leads to a weaker response to restraint stress and a strongly increased resistance to lipopolysaccharide-induced endotoxic shock. These results underscore the importance of tight regulation of glucocorticoid receptor expression for the control of physiological and pathological processes. Furthermore, they may explain differences in the susceptibility of humans to inflammatory diseases and stress, depending on individual prenatal and postnatal experiences known to influence the expression of the glucocorticoid receptor.

The WD40-domain containing protein CORO2B is specifically enriched in glomerular podocytes and regulates the ventral actin cytoskeleton.

Podocytes are highly specialized epithelial cells essentially required to establish and maintain the kidney filtration barrier. Due to their complex cellular architecture these cells rely on an elaborated cytoskeletal apparatus providing plasticity as well as adaptive adhesion properties to withstand significant physical filtration forces. However, our knowledge about podocyte specific components of the cytoskeletal machinery is still incomplete. Employing cross-analysis of various quantitative omics-data sets we identify the WD40-domain containing protein CORO2B as a podocyte enriched protein. Furthermore, we demonstrate the distinct localization pattern of CORO2B to the ventral actin cytoskeleton serving as a physical linkage module to cell-matrix adhesion sites. Analysis of a novel Coro2b knockout mouse revealed that CORO2B modulates stress response of podocytes in an experimental nephropathy model. Using quantitative focal adhesome proteomics we identify the recruitment of CFL1 via CORO2B to focal adhesions as an underlying mechanism. Thus, we describe CORO2B as a novel podocyte enriched protein influencing cytoskeletal plasticity and stress adaptation.

MOF maintains transcriptional programs regulating cellular stress response.

MOF (MYST1, KAT8) is the major H4K16 lysine acetyltransferase (KAT) in Drosophila and mammals and is essential for embryonic development. However, little is known regarding the role of MOF in specific cell lineages. Here we analyze the differential role of MOF in proliferating and terminally differentiated tissues at steady state and under stress conditions. In proliferating cells, MOF directly binds and maintains the expression of genes required for cell cycle progression. In contrast, MOF is dispensable for terminally differentiated, postmitotic glomerular podocytes under physiological conditions. However, in response to injury, MOF is absolutely critical for podocyte maintenance in vivo. Consistently, we detect defective nuclear, endoplasmic reticulum and Golgi structures, as well as presence of multivesicular bodies in vivo in podocytes lacking Mof following injury. Undertaking genome-wide expression analysis of podocytes, we uncover several MOF-regulated pathways required for stress response. We find that MOF, along with the members of the non-specific lethal but not the male-specific lethal complex, directly binds to genes encoding the lysosome, endocytosis and vacuole pathways, which are known regulators of podocyte maintenance. Thus, our work identifies MOF as a key regulator of cellular stress response in glomerular podocytes.

Differential expression of RNA and protein of the three pore-forming subunits of the amiloride-sensitive epithelial sodium channel in taste buds of the rat.

Salt taste signals from the rat anterior tongue are probably transduced via epithelial sodium channels (ENaCs) residing in the apical cellular pole of taste cells. The signals are blocked by mucosal amiloride in low microM concentrations. In contrast, the rat vallate papilla does not contribute to amiloride-blockable salt taste. Two approaches were used to probe for the three subunits of ENaC in the anterior and posterior tongue of the rats in sodium balance. (a) Immunohistochemistry with antibodies against ENaC subunits and against amiloride binding sites. In the anterior tongue, reactivity for alpha-, beta-, and gamma-subunits was present in taste buds and lingual epithelium. In the posterior tongue vallate papilla, reactivity for alpha-subunit and for amiloride binding sites was easily demonstrable, whereas that for beta-subunit and especially for gamma-subunit was weaker than in the anterior tongue. (b) RT-PCR techniques were used to probe for the presence of ENaC subunit mRNA. In isolated taste buds of the anterior tongue, mRNA of all three subunits was found, whereas in isolated taste buds of the vallate papilla only mRNA of the alpha-subunit was easily detectable. That of beta- and gamma-subunits was much less abundant. RNA of all three subunits was abundant only in taste buds of the anterior tongue. Therefore, subsets of elongated taste cells do express ENaC, but regional differences exist in the transcription and expression of subunits. The regional differences suggest that amiloride-sensitive salt taste, which requires all three subunits, is present in the anterior but not the posterior tongue of rats, as functional studies indicate.

Occurrence of ENaC subunit mRNA and immunocytochemistry of the channel subunits in taste buds of the rat vallate papilla.

Epithelial Na+ channels (ENaCs) are thought to mediate the amiloride-blockable salt taste. The rat vallate papilla does not contribute to amiloride-blockable salt taste, yet the presence of ENaC-mRNA in this tissue has been reported. Is ENaC actually contained in the taste cells, or is it merely present in the supporting lingual epithelium? To avoid contamination by ENaC contained in the lingual epithelium, we physically isolated taste buds from the vallate papilla and used mRNA purification followed by reverse transcriptase polymerase chain reaction (RT-PCR) to investigate the presence of ENaC-type message in the isolated buds. mRNA of alpha-, beta- and gamma-subunits was detected, the alpha-signal being the strongest. These results provide first molecular evidence for the presence of ENaC subunits in taste buds that were isolated from the posterior tongue and were free of epithelial contamination. In addition, we used immunohistochemistry to show ENaC-like reactivity in posterior tongue taste cells. Interestingly, the immunoreactivity was not predominantly apical but was intracellular and close to or at the basolateral membrane. The function of basolateral ENaC-type channels is unknown. Possibly, the channels are normally closed or of very low open probability in the resting state.

The mineralocorticoid receptor expression in the mouse CNS is conserved during development.

Using a specific polyclonal antiserum raised in rabbit against amino acids 1-23 of the mouse mineralocorticoid receptor (MR) we investigated the developmental profile of MR expression in the murine CNS by immunocytochemistry. MR protein appeared first at embryonic day E16.5 in the limbic system, i.e. in the hippocampus and induseum griseum. During development and in adulthood, high levels of MR expression were observed in the limbic system, whereas expression levels detectable in layers II, III, V of the neocortex and in motoneurons of cranial nerves and spinal cord were lower. No MR staining was found in the hypothalamus. Developmental MR expression was restricted to neuronal populations that also express MR protein in the adult CNS, indicating that the MR may fulfill the same functions in neurons during development and in adulthood.

Corticotropin-releasing hormone expression is the major target for glucocorticoid feedback-control at the hypothalamic level.

Glucocorticoid production is controlled via the hypothalamo-pituitary-adrenal (HPA) axis by a negative feedback mechanism involving the glucocorticoid receptor (GR). A major site of regulation is the hypothalamus, where the GR is thought to repress the expression of genes such as corticotropin-releasing hormone (CRH) and arginine-vasopressin (AVP). To define the role of the GR in this feedback loop in more detail, the content of CRH, AVP and neurophysin in the median eminence of mice carrying a targeted disruption of the GR gene was studied using immunohistochemistry. GR-deficient mice were found to contain five times more CRH in the median eminence than wild-type littermates. In contrast, no significant change in the content of AVP was observed in the outer layer of the median eminence and neurophysin was also only moderately increased. Our studies suggest that, at the hypothalamic level, CRH synthesis is the major target for feedback control by the GR and that transcriptional control of AVP and neurophysin plays only a supportive role in this process.

Oral naloxone reduces constipation but not antinociception from oral morphine in the rat.

Oral administration of naloxone (10 mg/kg) antagonized the slowing of the intestinal transit caused by oral morphine (1, 2.5 and 5 mg/kg) in rats. Oral administration of naloxone (10 mg/kg) did not prevent the antinociceptive effect of orally administered morphine (2.5 mg/kg) in the tail-flick test carried out on rats. It is concluded that oral naloxone locally blocks the constipating effect of morphine, while it fails to reduce the central action of morphine due to extensive metabolization after oral administration.

Coincubation of tissue slices, a new way to study metabolic cooperation between organs: hepatorenal cooperation in the biotransformation of CGP 47 969 A.

INTRODUCTION: There is limited information on in vitro/ex vivo tools to be used for studying interorgan metabolic cooperation. We report here the use of the tissue slice technique for this purpose. METHODS: Rat liver and kidney slices were used to study metabolic cooperation for the metabolism of CGP 47 969, a potential anti-inflammatory compound which in vivo is extensively conjugated with glutathione and subsequently degraded via the mercapturic acid pathway. RESULTS: Upon incubation with liver slices, CGP 47 969 was extensively conjugated with GSH whilst degradation of the GSH conjugate was moderate. Upon incubation with kidney slices, conjugation of CGP 47 969 with GSH was moderate but degradation of the GSH conjugate was complete. Upon coincubation of CGP 47 969 with liver and kidney slices, both conjugation with GSH and its subsequent degradation were almost complete. Thus, coincubation of liver and kidney slices permitted the efficient in vitro reproduction of the complete biotransformation of CGP 47 969 via its GSH conjugate to the ultimate mercapturic acid metabolite in a one step procedure. DISCUSSION: This novel slice coincubation culture could serve as an in vitro model for interorgan cooperation in multistep metabolic processing.

Characterization of bolesatine, a toxic protein from the mushroom Boletus satanas Lenz and it's effects on kidney cells.

Protein, DNA, and RNA syntheses were assayed in Madin Darby canine kidney cells (MDCK) treated by bolestaine, a toxic glycoprotein from the mushroom Boletus satanas (single chain, Mr 63,000 +/- 3000, pI 8.3 +/- 0.1, disulphide intrachain bridge) previously shown to be an inhibitor of in vitro protein synthesis. Cellular protein and DNA syntheses are inhibited in a dose-dependent manner after 24 h of incubation, whereas the uptake of the labelled precursors of proteins, DNA and RNA biosyntheses into the cells is not affected. The IC50 of bolesatine for protein synthesis is 0.14 microM in the cell culture medium. RNA synthesis is not inhibited at this concentration. The IC50 for DNA synthesis is 0.32 microM. When galactose is added to the culture medium, it decreases or even abolishes the toxic effects, indicating that it prevents the toxin from binding on the membrane and penetrating inside the cells. The profiles of polysomes in MDCK cells treated with bolesatine, compared to the untreated ones, show an increasing pool of polysomes indicating that the toxin acts on the peptidyl elongation step in the protein synthesis.

Inhibition of protein synthesis in liver and kidney of mice by bolesatine: mechanistic approaches to the mode of action at the molecular level.

Protein synthesis was assayed in liver and kidney of mice treated with bolesatine, a toxic glycoprotein from the mushroom Boletus satanas (Lenz) which was previously shown to be an inhibitor of protein synthesis by cell-free systems in vitro and by cultured cell-lines. Protein synthesis in vivo (Swiss mice) is inhibited in a dose-dependent manner in liver and kidney. The mechanism of action does not appear to be due to RNA-N-glycosidase activity of bolesatine or a RNAase activity of this toxin on the ribosomal RNAs. Ribosomes do not appear to be damaged by pretreatment with bolesatine as judged by a poly(U) translation system. Thus bolesatine cannot be included in the group of protein synthesis inhibitors of plant origin known as ribosome-inactivating proteins (RIPs).

Effect of bolesatine, a glycoprotein from Boletus satanas, on rat thymus in vivo.

Bolesatine is a glycoprotein purified to homogeneity from Boletus satanas Lenz, a toxic mushroom which causes serious gastroenteritis. This lectin possesses a mitogenic activity on human lymphocytes at very low concentrations, whereas higher concentrations inhibit protein synthesis in vitro in several systems. The mitogenic activity on peripheral blood T lymphocytes in vitro has been shown to be at least 200-fold higher than the activity of the well studied phytohemagglutinin (PHA). In order to verify this property in vivo, the effect of bolesatine has been studied in thymus of rats given orally bolesatine. Two groups of bolesatine-treated animals were used in addition to the control group. One group was given every 48 h, 28 micrograms of bolesatine/kg body weight seven times and 150 micrograms/kg body weight 48 h before the sacrifice. The other group was given 55 micrograms of bolesatine/kg body weight according to the same protocol and 150 micrograms/kg body weight 48 h before the sacrifice. In these conditions, the ratio thymus weight/body weight is increased by 10% and 28%, respectively, in groups 1 and 2. Similarly, the DNA synthesis is increased by more than 50%, indicating that (i) bolesatine probably possesses a mitogenic effect on thymocytes in vivo (ii) that the increase of the ratio thymus weight/body weight is not due to swelling by water retention, but rather to a multiplication of thymocytes. These results are confirmed in a second run of experiments in which bolesatine given orally to rats in lower doses of 3-12 micrograms/kg induces an increase of both thymus weight by 47% to 54% and an increase of total proteins by 52% to 56%, respectively, whereas the ratio total protein/g of thymus does not change. Thus bolesatine, known to be mitogenic to human lymphocytes in vitro is also mitogenic to rat thymocytes in vivo.

Effects of bolesatine on a cell line from the SP2/O thymic lymphosarcoma.

Bolesatine, a toxic protein isolated from Boletus satanas Lenz inhibits in vitro protein synthesis in a concentration-dependent manner in a cell line from a radiation-induced thymic lymphosarcoma (SP2/O) with a 50% inhibitory concentration (IC50) of 9.5 nM (0.6 microgram/ml). In vivo, an i.p. single injection of bolesatine, corresponding to 1/6 and 1/10 of 24-h 50% lethal dose, in Balb/c mice having ascitic tumour induced by the i.p. preinjection of SP2/O cells allows a remission of 50% and 30%, respectively. Treated mice survived 120 days after the treatment, i.e. 90 days after the death of control animals.

Properties of bolesatine, a translational inhibitor from Boletus satanas Lenz. Amino-terminal sequence determination and inhibition of rat mitochondrial protein synthesis.

Bolesatine is a toxic monomeric glycoprotein of Mr 63,000 isolated from the mushroom Boletus satanas Lenz. Its N-terminal amino acid sequence was established: NH2-Thr-Trp-Arg-Ile-Tyr-Leu-Asn-Asn-Gln-Thr-Val-Lys-Leu-Ala-Leu-Leu-Leu- Pro- Asn-Gly.... It inhibits protein synthesis in isolated rat mitochondria. After 90 min of preincubation with bolesatine, the incorporation of [14C]-leucine into mitochondrial proteins was inhibited with an IC50 of 530 nM.

Bolesatine induces agglutination of rat platelets and human erythrocytes and platelets in vitro.

Bolesatine is a toxic glycoprotein isolated from the mushroom Boletus satanas Lenz, which has been shown to inhibit protein synthesis in cell-free systems and cell culture. It is toxic to rodents, the LD50% 24 h being 1 mg kg-1 (i.p.) and 0.15 mg kg-1 (i.v.) in the rat in which it induces hepatic blood stasis. Bolesatine possesses lectinic properties with in particular a sugar binding site for D-galactose and mitogenic activity toward lymphocytes. Tested for cell agglutination on red blood cells and platelets, bolesatine agglutinates both human and rat platelets from threshold concentrations of 30 and 300 nM respectively. EDTA and PGI2 (aggregation inhibitors) do not decrease the agglutination induced by bolesatine, indicating that the process does not involve platelet activation. In contrast, fibrinogen decreases platelet agglutination induced by bolesatine, most likely by masking the binding sites on platelets or by interacting with the toxin. Bolesatine agglutinates all red blood cells without any blood group specificity in the concentration range of 20 to 40 nM. This haemagglutination cannot be prevented by sugars, including D-galactose at a concentration of 0.5 M.

Aspirin and heparin prevent hepatic blood stasis and thrombosis induced by the toxic glycoprotein Bolesatine in mice.

Bolesatine is a toxic glycoprotein isolated from Boletus satanas Lenz, which inhibits protein synthesis in vivo and in vitro. The LD50 (24 h) is 1 mg /kg bw (i.p.), in mice and rats. When given i.p. to mice (0.1 - 1.0 mg/kg bw) bolesatine induced thrombi and blood stasis in the liver, 5 - 21 h after injection, and modifications of the number of blood corpuscles in peripheral blood. These effects were efficiently reversed by aspirin, ticlopidin and heparin (as attested by histology and electron microscopy) which however failed to prevent death in animals given lethal doses. Together, these results showed that the death of bolesatine poisoned animals given high doses, was rather due to a combination of thrombosis and other toxic effects. In addition, they suggest that these antithrombotic drugs may overcome cases of human poisoning, with low exposures of this boletus, showing a hypertension probably due to mechanical obstruction which resists normal therapy.

Comparative pharmacokinetics of RAD001 (everolimus) in normal and tumor-bearing rodents.

PURPOSE: Comparative pharmacokinetic (PK) analysis of the mTOR inhibitor RAD001 (everolimus) in rats and mice. METHODS: Blood cell partitioning, plasma protein binding and PK parameters of RAD001 in blood and tissues (including brain) of both mice and rats were determined. PK modeling predicted plasma/blood and tumor levels from a variety of regimens and these were compared with the known human PK profile. DCE-MRI was used to compare tumor vascularity between mice and rats. Estimation of IC50 values in vitro and ED50 values in vivo were used to provide an indication of anti-tumor activity. RESULTS: The PK properties of RAD001 differed between mice and rats, including erythrocyte partitioning, plasma protein binding, plasma/blood t(1/2), oral bioavailability, volume of distribution, tissue/tumor penetration and elimination. Modeling of tumor and blood/plasma PK suggested that in mice, multiple daily administrations result in a 2-fold increase in tumor levels of RAD001 at steady state, whereas in rats, a 7.9-fold increase would occur. Weekly high-dose regimens were predicted not to facilitate tumor accumulation in either species. Total tumor levels of RAD001 were four- to eight-fold greater in rats than in mice. Rat tumors had a >2-fold greater plasma content and permeability compared to mouse tumors, which could contribute to differences in tumor drug uptake. Maximal antitumor effects (T/C of 0.04-0.35) were observed in both species after daily administration with similar C(max) and AUC values of unbound (free) RAD001. These free levels of RAD001 are exceeded in serum from cancer patients receiving clinically beneficial daily regimens. In rodents, brain penetration of RAD001 was poor, but was dose-dependent and showed over-proportional uptake in rats with a longer t(1/2) compared to the systemic circulation. CONCLUSIONS: The PK of RAD001 differed between mice and rats, with rats having a PK profile closer to that of humans. High intermittent doses of RAD001 may be more appropriate for treatment of brain tumors.

Disposition of the toxic protein, bolesatine, in rats: its resistance to proteolytic enzymes.

1. Bolesatine is a toxic protein (LD50 oral 3.3 mg/kg in mice) isolated from the mushroom Boletus satanas Lenz, which inhibits protein synthesis in vitro. It induces gastroenteritis in human. 2. 14C-Bolesatine, given orally to rats (30 micrograms/kg), is distributed in the gastrointestinal, tract, kidney, liver and, to a lesser extent, in the thymus, spleen and lung. Bolesatine is eliminated in faeces and urine (80% in 24h). 3. The material excreted in urine is not proteolysed, and no protease (trypsin, chymotrypsin, pronase, proteinase K, Staphylococcus aureus (strain V8) protease and pepsin) is found to hydrolyse bolesatine in either its native or denatured form. However, thermolysin hydrolysed denatured bolesatine to a protein having a Mr of about 55 kD. 4. Bolesatine is found in all the following rat liver and kidney subcellular fractions: cytoplasm, mitochondria, ribosomes, microsomes and nuclei.

Effect of bolesatine on phospholipid/calcium dependent protein kinase in Vero cells and in rat thymus.

Bolesatine, a glycoprotein from Boletus satanas Lenz, has previously been shown to be mitogenic to rat and human lymphocytes at very low concentrations, whereas higher concentrations inhibit protein synthesis in vitro and in several in vivo systems. The mechanism whereby this mitogenic activity occurs was previously unknown. To elucidate this mechanism, the effects of bolesatine have been studied in a cell-free system, VERO cells, and in vivo in rat thymus. In a cell-free system, bolesatine appears to be a direct effector of PKC. The activation is concentration dependent for 1-10 ng/ml. At the same time, VERO cells significantly proliferate when incubated with the bolesatine (3, 5 and 10 ng/ml), since the DNA synthesis increases by 27, 48, and 59%, for respectively, 3, 5 and 10 ng/ml compared with control. Moreover, Bolesatine (5 and 10 ng/ml) induces InsP3 release in a concentration-dependent manner (114 and 142%) as compared to control. In vivo, 24 h after oral administration of bolesatine to rates (20, 100 and 200 microg/kg), PKC activity is significantly increased in thymus. THe most effective doses (100 and 200 microg/kg) give 590-620% increase in cytosolic PKC activity and 85-91% increase in total PKC activity as compared to control. This PKC activation by bolesatine in rat thymus is directly linked to the mitogenic activity observed in vivo. Bolesatine is thus capable of activating the PKC directly and/or indirectly (via InsP3 release) during its mitogenic processes.