Increased expression of the mineralocorticoid receptor in the brain of spontaneously hypertensive rats.

The mineralocorticoid receptor (MR) has been considered as both neuroprotective and damaging to the function of the central nervous system. MR may be also involved in central regulation of blood pressure. In the present study, we compared the expression of MR and the glucocorticoid receptor (GR) in the hippocampus and hypothalamus of 16-week-old spontaneously hypertensive rats (SHR) and normotensive control Wistar Kyoto (WKY) rats. In the hippocampus, MR expression was studied by in situ hybridization (ISH), quantitative polymerase chain reaction (PCR) and immunohistochemistry, whereas GR expression was analysed using the latter two procedures. Hypertensive animals showed an increased expression of MR mRNA in the whole hippocampus according to qPCR data and also in CA3 by ISH. Immunocytochemical staining for MR of the dorsal hippocampus, however, did not reveal differences between SHR and WKY rats. SHR showed elevated hypothalamic MR mRNA by qPCR, as well as an increased number of MR immunopositive cells in the magnocellular paraventricular region, compared to WKY rats. By contrast, expression levels of GR mRNA or protein in the hippocampus and hypothalamus of SHR were similar to those of WKY rats. Furthermore, we investigated the role of MR in the hypertensive rats by i.c.v. injection of the MR antagonist RU-2831. This compound produced a significant drop in blood pressure for SHR. In conclusion, MR expression is increased in the hippocampus and hypothalamus of SHR. We suggest that pathological MR overdrive may take responsibility for up-regulation of blood pressure and the encephalopathy of hypertension.

Oxygen transfer coefficient and the kinetic parameters of exo-polygalacturonase production by Aspergillus flavipes FP-500 in shake flasks and bioreactor.

AIMS: To evaluate and compare the sensitivity of Exo-PG production and kinetic parameters of Aspergillus flavipes FP-500 to oxygen transfer condition in shake flasks and bioreactor. METHODS AND RESULTS: Aspergillus flavipes FP-500 was grown on pectin as carbon source in shake flasks and bioreactor at different oxygen transfer conditions. The volumetric coefficient of oxygen transfer (kLa) was modified by changing both, the flask size/medium volume ratio and the agitation speed. Higher biomass concentration, Exo-PG activity, maximum specific growth rate and yield coefficient were obtained in bioreactor at higher kLa value. A strong correlation was found between biomass, Exo-PG activity and growth-associated product coefficient to kLa in bioreactor but does not in shake flasks. The mathematical model provided a good description of growth, pectin consumption and Exo-PG production in submerged batch cultures carried out in bioreactor. CONCLUSIONS: Biomass concentration, Exo-PG activity and their kinetics of Aspergillus flavipes FP-500 were strongly influenced by oxygen transfer condition and cultivation system. Significance and Impact of Study The production of enzymes by fungal fermentation is strictly aerobic and understanding the influence of oxygen transfer condition on the production kinetic is of vital importance in order to design, optimize and translate bioprocesses to industrial scale.

Progress in primary aldosteronism: present challenges and perspectives.

Primary Aldosteronism (PA) is a disorder of the adrenal zona glomerulosa (ZG) in which aldosterone secretion is increased and is relatively autonomous of normal regulatory mechanisms. A recent conference in Munich organized by Prof. Reincke addressed advances and challenges related to the screening, diagnosis, and identification of uni- and bilateral involvement of the diseased adrenal of PA. Some infrequently addressed issues are described herein. We postulate that most cases of PA are due to the activation by unknown mechanisms of subset of cells resulting in the formation of a multiple foci or nodules of hyperactive zona glomerulosa cells. This implies that one or several yet unidentified stimuli can drive aldosterone overproduction, as well as the proliferation of aldosterone-producing cells. Current diagnostic procedures allow to determine whether inappropriate aldosterone production is driven by one or both adrenal glands and thus to establish optimal treatment.

A highly sensitive immunofluorometric assay for the measurement of aldosterone in small sample volumes: validation in mouse serum.

Data on the involvement of aldosterone in the regulation of the renin-angiotensin-aldosterone system (RAAS) in rodents are still scarce, partly due to the high sample volumes needed by commercially available assays and to the very low aldosterone concentrations present. We have developed a highly sensitive and non-isotopic immunoassay, requiring a volume of only 50 microl serum for a duplicate measurement, employing a highly specific monoclonal antibody against aldosterone. The assay was validated in human and mouse samples and exhibited a linear working range from 10 to 1000 pg/ml. Values obtained after a chromatographic purification step correlated significantly to the dichloromethane extraction ordinarily used. Basal aldosterone values were measured in 75 mouse hybrids and found within the linear range (173+/-21 pg/ml), with no significant difference between males and females. Additionally, we show an increase in serum aldosterone in mice from 3 to 11 weeks of age. Mice of the same genetic background were treated with dexamethasone intraperitoneally (n=7), resulting in significantly decreased concentrations (35+/-3 vs 114+/-33 pg/ml in controls; P<0.001). In contrast, adrenocorticotropic hormone resulted in significantly increased serum aldosterone (603+/-119 pg/ml; n=7; P<0.001), as did the physiological stimulation of the RAAS by a high K(+)/low Na(+) diet (1369+/-703 vs 172+/-36 pg/ml). In conclusion, we have developed and validated an extremely sensitive assay for determination of aldosterone concentrations from very small serum samples, which could be especially useful in pharmacological intervention studies in rodent models.

11beta-hydroxylase and aldosterone synthase expression in fetal rat hippocampal neurons.

The central nervous system produces many of the enzymes responsible for corticosteroid synthesis. A model system to study the regulation of this local system would be valuable. Previously, we have shown that primary cultures of hippocampal neurons isolated from the fetal rat can perform the biochemical reactions associated with the enzymes 11beta-hydroxylase and aldosterone synthase. Here, we demonstrate directly that these enzymes are present within primary cultures of fetal rat hippocampal neurons.

The 11beta hydroxysteroid dehydrogenase 2 exists as an inactive dimer.

The 11beta-hydroxysteroid dehydrogenase types 1 and 2 enzymes (11beta-HSD1 and 11beta-HSD2), modulate glucocorticoid occupation of the mineralocorticoid and glucocorticoid receptors by interconverting corticosterone and cortisol to the inactive metabolites 11-dehydrocorticosterone and cortisone within the target cells. The NAD(+)-dependent 11-HSD 2 in the kidney inactivates corticosterone and cortisol, allowing aldosterone, which is not metabolized, access to the receptor. Studies of the kinetics of 11-HSD 2 activity in the rat kidney have produced inconsistent results. Western blots done in the absence of the reducing agent beta-mercaptoethanol showed two bands with approximate MW of 40 and 80 kDa. When beta-mercaptoethanol was used, only the 40 kDa was detected, indicating that under non-denaturing conditions a significant proportion of the 11beta-HSD 2 exists as a dimer. NAD(+)-dependent conversion of 3H-corticosterone by 20 microg of microsomal protein increased approximately 10 fold with the addition of 5 mM DTT concentration. NADP(+)-dependent activity with 20 microg of microsomal protein was very low and did not change significantly when using DTT. In the presence of DTT, the predominant 11-HSD activity in the rat kidney is NAD(+)-dependent with a K(m) of 15.1 nM, similar to that of the cloned and expressed enzyme. These data suggest that dimerization and subsequent enzyme inactivation occur when protocols promoting oxidation of this protein are used.

Steroidogenesis in the human skin: 21-hydroxylation in cultured keratinocytes.

We have evaluated the metabolism of radiolabeled progesterone (P) by the microsomal fraction isolated from HaCaT keratinocytes. P was widely metabolized to different compounds that included DOC (5-7% conversion) thus demonstrated 21-hydroxylase (21-OHase) activity, a key step in adrenal synthesis of gluco- and mineralocorticoids. However, RT-PCR amplification for the CYPc21 transcript of the corresponding gene showed no evidence for gene expression in HaCaT cells suggesting that the 21-OHase enzyme present in keratinocytes is different from that described in adrenal gland. Further characterization showed that whereas estradiol stimulated markedly P metabolism by HaCaT microsomes, with generation of new unidentified compounds, Lineweaver-Burk analysis of keratinocyte 21-OHase activity showed that the K(m) and V(max) were unaffected by estrogen. The apparent K(m) was 0.6 microM without estradiol and 0.7 microM with estradiol, while the respective V(max) values were 60 and 76 nmol/l/min. To conclude, we found extensive metabolism of P in human keratinocytes, we also provide the first demonstration of 21-OHase activity in this cell system and further showed that it is coded by a gene different from the adrenal CYPc21.

Serum 18-hydroxycortisol in primary aldosteronism, hypertension, and normotensives.

This study reports the determination of plasma 18-hydroxycortisol (18-OHF) using a new and easy enzyme-linked immunosorbent assay (ELISA) method in primary aldosteronism and compares the values found in essential hypertensives and normotensive controls. In primary aldosteronism, we evaluated usefulness of plasma 18-OHF determination and the dexamethasone suppression test in the diagnosis of glucocorticoid-remediable aldosteronism using the genetic test as the gold standard. We studied 31 primary aldosteronism patients, 101 essential hypertensives, and 102 healthy normotensive controls. The plasma 18-OHF was measured using a biotin-avidin enzyme-linked assay by a new and purified polyclonal antibody. The 18-OHF value in primary aldosteronism was 6.3+/-8.05 nmol/L; this value is significantly higher than the value found in essential hypertensives and normotensive controls (2.81+/-1.42 and 2.70+/-1.41 nmol/L, respectively; P<0.0005). In primary aldosteronism, 4 of 31 patients had 18-OHF levels that were 10 times higher than the normal upper limit (2.983 nmol/L). The dexamethasone suppression test in primary aldosteronism patients was positive (serum aldosterone <4 ng/dL) in 13 of 31 cases. A chimeric CYP11B1/CYP11B2 gene was demonstrated in 4 primary aldosteronism patients, corresponding to the same cases that had higher level of 18-OHF. In conclusion, plasma 18-OHF determination by this ELISA method is reliable for detecting glucocorticoid-remediable aldosteronism, and it does so better than the dexamethasone suppression test.

Aldosterone esters and the heart.

There are clinical and experimental situations in which symptoms of mineralocorticoid excess are remediable with mineralocorticoid receptor antagonist treatment, in spite of paradoxically low levels of plasma renin and aldosterone. Several decades ago, a factor isolated from the heart was described that had mineralocorticoid properties like those of aldosterone, but much more potent. It was thought to be similar to aldosterone-18-monoacetate or -21-monoacetate, acetyl derivatives of aldosterone that are very rapidly hydrolyzed in the circulation. In our efforts to confirm and extend these observations, we extracted rat hearts and plasma harvested in a manner that would minimize hydrolysis. The product was subjected to several forms of TLC and HPLC and compared to several acetylated derivatives of aldosterone standards. We found that 68% of the aldosterone extracted from fresh myocardium corresponded to an aldosterone derivative that migrates at the same rate as aldosterone-20-monoacetate. The identity of this compound awaits definitive analysis. Tritiated aldosterone-21-monoacetate hydrolyzed to form aldosterone very rapidly; negligible monoacetate remained in blood left at 37 degrees C for 5 min or in hearts left at room temperature for 30 min. Regulation of aldosterone production serves the requirements of fluid and electrolyte homeostasis provided by transport epithelia, primarily that of the kidney. Nonepithelial actions of aldosterone would be freed of these regulatory constraints if the formation of a more potent derivative of the parent compound to which it is almost immediately hydrolyzed in the circulation were regulated within the nonepithelial target tissues.

Role of central mineralocorticoid receptors in cardiovascular disease.

Mineralocorticoids act directly through their receptors in specific centers in the central nervous system, kidneys, heart, and vascular smooth muscle to mediate hemodynamic homeostasis. These steroids also modulate renal and cardiovascular function indirectly through the autonomic nervous system. Complex homeostatic mechanisms under normal hormonal control become pathogenic when there is an excess of regulatory hormone. Experiments in which mineralocorticoid receptor antagonists or antisense oligodeoxynucleotides were administered centrally have clearly shown that centrally mediated effects on salt appetite, baroreceptor function, and autonomic drive to the renal and cardiovascular systems are crucial to the pathogenesis of hypertension and cardiovascular disease of hyperaldosteronism, and certain forms of genetic hypertension.

Angiotensin stimulates the expression of interferon-inducible genes in H295R cells.

Angiotensin-II (A-II) induces proliferation of zona glomerulosa cells and stimulates expression of cytochrome P-450 aldosterone synthase. The genes activated during this adrenal remodeling are not well defined. To clarify this mechanism, we sought to identify the genes whose expression is stimulated by A-II in the H295R cell line. Using a subtractive hybridization technique, we identified one clone whose expression was stimulated by A-II. The sequence of this gene was homologous to the human interferon-inducible genes, 9-27, 1-8D and 1-8U. The 5' portion of the gene was identical to the 1-8D gene product and the 3' was identical to the 9-27 gene product, but the existence of a transcript was not demonstrated by RT-PCR. The expression of these three genes was stimulated by A-II, with the 9-27 gene being most abundant. Potassium and forskolin also stimulated the expression of the 9-27 gene in the H295R cells, but not as effectively as did A-II or interferon-gamma.

Inhibition of aldosterone production in rat adrenal mitochondria by 18-ethynyl-11-deoxycorticosterone: a simple model for kinetic interpretation of mechanism-based inhibitors.

A simple mathematical model for studying mechanism-based inhibitors (MBIs) is presented. The mathematical equations are deduced for an experimental protocol consisting of a first incubation of the enzyme in the presence of MBI followed by a washing protocol to eliminate free MBI. Finally enzyme activity (initial velocity) is measured with specific substrate. The representation of the final equation obtained is a straight line, and the MBI-specific association constant of velocity (k) can be calculated from its slope. The mathematical model was then challenged with the effect of 18-ethynyl-11-deoxycorticosterone (18-EtDOC) as an MBI on aldosterone biosynthesis from 11-deoxycorticosterone (DOC) in rat adrenal mitochondria. The last step of the mitochondrial biosynthesis of aldosterone consists of the conversion of DOC into corticosterone (B) or 18-hydroxy-11-deoxycorticosterone (18-OHDOC), and both steroids can then be transformed into aldosterone. The k (mM(-1) x min(-1)) values obtained for 18-EtDOC were: 451 +/- 36 for DOC to aldosterone; 177 +/- 16 for B to aldosterone; 175 +/- 15 for 18-OHDOC to aldosterone; and 2.7 +/- 0.2 for DOC to B. These results show that this MBI practically does not affect the metabolism of DOC to B in our enzyme preparation and that conversions of B and 18-OHDOC into aldosterone are catalyzed by the same enzyme.

Expression of 11beta-hydroxylase and aldosterone synthase genes in the rat brain.

The terminal stages of cortisol and aldosterone production in the human adrenal gland are catalysed by the enzymes 11beta-hydroxylase and aldosterone synthase, which are encoded by the CYP11B1 and CYP11B2 genes respectively. Recent studies have suggested that aldosterone and cortisol are also made in other tissues such as the brain, heart and vascular system and may play a role in cardiovascular homeostasis. The aim of this study was to confirm the presence of these enzymes and localise them precisely in the rat brain. Reverse transcription-polymerase chain reaction (RT-PCR)/Southern blotting confirmed transcription of CYP11B1 and CYP11B2 in whole brain and hypothalamus minces from Wistar-Kyoto rats. 11beta-Hydroxylase and aldosterone synthase were immunolocalised in paraffin-embedded rat adrenal and brain sections using mouse monoclonal antibodies. Negative controls utilised a mouse monoclonal antibody raised against a non-mammalian epitope. In the brain, 11beta-hydroxylase and aldosterone synthase were detected in the cerebellum, especially the Purkinje cells, as well as the hippocampus. The specificities of the 11beta-hydroxylase and aldosterone synthase antibodies were confirmed by positive immunostaining of the relevant regions of the adrenal cortex. This is the first direct evidence that steroid hydroxylases involved in the final stages of corticosteroid biosynthesis are present in specific regions of the central nervous system.

Cloning and expression of the bovine 11beta-hydroxysteroid dehydrogenase type-2.

The bovine 11beta-hydroxysteroid dehydrogenase type 2 enzyme (11beta-HSD-2) cDNA was cloned from three overlapping PCR fragments using primers based on the human and ovine 11beta-HSD-2 cDNA sequences. Both cDNA ends were obtained by a modified RACE (Rapid Amplification of cDNA Ends) method. The bovine 11beta-HSD-2 cDNA is 1878 bp long, excluding the poly(A) tail. It consists of a 5'-untranslated region of 133 bp, an open reading frame of 1215 bp and a 3'-untranslated region of 530 bp. Bovine 11beta-HSD-2 cDNA is highly homologous to that of the sheep (92%) and less related to the human (67%), rabbit (65%), rat (52%) and mouse (45%) cDNA. The predicted bovine 11beta-HSD-2 protein contains 404 amino acid residues with a calculated mol wt of 43,985. It is homologous to the sheep (98%) and human (88%) protein, and less related to that of the rabbit (76%), rat (80%) and mouse (77%). The cloned 11beta-HSD-2 cDNA was transfected into CHOP cells and the enzymatic characteristics determined. The enzyme functions primarily as an oxidase, uses NAD(+) and is more active with corticosterone as a substrate than with cortisol or dexamethasone. It is expressed in high concentrations in kidney, adrenal and colon, and in small concentrations in liver, heart and lung. In conclusion, the 11beta-HSD-2 enzyme of cattle is very similar to that of other species in its structure and enzymatic characteristics.

The effect of chronic food and water restriction on open-field behaviour and serum corticosterone levels in rats.

In operant conditioning experiments, two methods are commonly used to motivate laboratory rats to perform designated tasks. The first is restricting food so that rats are forced to lose 20% of body weight within one week, followed by maintenance at 80% of the baseline weight for the remainder of the experiment. The second is restricting access to water to 15 min in each 24 h period. These methods are effective in motivating the animals. There is, however, little information available on the effects on performance in tests of behaviour that are not related to operant conditioning. In addition, it is not clear if these commonly used methods of food and water restriction will lead to physiological stress as indicated by an elevation of serum corticosterone. Male rats were either food-restricted to reduce and maintain their weight at 80% of baseline weight, or were restricted to 15 min access to water every 24 h. Activity in the open field was significantly greater in food-restricted rats than in water-restricted or control rats, but freezing behaviour was similar in all experimental groups. Food-restricted rats had a higher mean serum corticosterone level than water-restricted and control rats 37 days after the start of the experimental period. These data suggested that chronically restricting food and maintenance of body weight at 80% of baseline body weight led to significant behavioural changes and physiological stress. In contrast, water restriction did not lead to changes in behaviour or corticosterone levels. A second experiment was conducted to compare the effects of food restriction to 80% of baseline body weight, as described above, with a less stringent protocol in which test rats were initially reduced to 80% of baseline weight, but were then maintained at 80% of an ad libitum fed control rat's weight. Serum corticosterone levels and adrenal gland weights were measured after the initial week of forced weight loss and after maintenance for 21 days. Forced loss of 20% of body weight in the first week led to significantly increased serum corticosterone levels and adrenal gland weights compared to ad libitum fed controls. Serum corticosterone levels and adrenal gland weights in rats maintained at 80% of their initial body weight for 21 days remained higher than ad libitum fed control rats. However, rats maintained at 80% of an ad libitum fed control rat's weight did not differ from control rats in serum corticosterone levels or adrenal gland weights at the end of the 21-day study period. Adjustment of the feeding regimen in this manner eliminated physiological evidence of chronic stress.

Active steroidogenesis in the normal rat skin.

Using the radiolabeled precursors of adrenal steroids (14)C-11-deoxycorticosterone (DOC) and (14)C-progesterone ((14)C-PROG) we demonstrate that rat skin can synthesize a number of steroids. TLC separation of labeled metabolites show that among the (14)C-steroid products, two co-migrate with corticosterone (B) and 11-dehydrocorticosterone (A) standards. Thus, normal rodent skin possesses steroidogenic activity that can be shown using progesterone or DOC as primary substrates.

An alternatively spliced rat mineralocorticoid receptor mRNA causing truncation of the steroid binding domain.

We attempted to clone the putative 11-dehydrocorticosterone receptor by RT-PCR with two degenerate primers from highly homologous regions of the DNA and steroid binding domains of the receptor subfamily. In doing so, we have identified an alternatively spliced variant mRNA of the rat mineralocorticoid (MR) with a ten bp deletion in the C-terminal steroid binding domain. This deletion results in a truncated MR receptor of 807 amino acids in comparison to the wild type of 981 amino acids. The deletion variant was expressed in colon, kidney, heart, liver, aorta and brain tissues. The relative abundance of the deletion variant compared to the wild type MR was estimated to be 6% in rat kidney and 4% in hippocampus. This deletion was also detected in human kidney by RT-PCR. Site-directed mutagenesis was used to create the eukaryotic expression plasmid pCR3-rMRdel10 from the wild type for a transactivation assay using the luciferase reporter system in CV-1 cells. The deletion variant had the same baseline transactivation activity as the wild type MR, but did not respond to aldosterone or corticosterone stimulation. Co-transfection of MR with the deletion variant had no significant effect on transactivation activity of the MR, indicating that the deletion variant is unlikely to serve as a negative regulator of MR function.

Corticosteroid production by fetal rat hippocampal neurons.

11beta-hydroxylase and aldosterone synthase catalyse the final stages of corticosterone and aldosterone synthesis respectively. Previously, we established that they are expressed in the rat brain, particularly the cerebellum and the hippocampus. Primary cultures of fetal rat neurons were studied. RT-PCR and immunohistochemistry established that neurons express 11beta-hydroxylase and aldosterone synthase mRNAs and protein. After incubating the cells with 10microM DOC for 24 hours, medium was analysed for aldosterone and corticosterone. Median % conversion of DOC to corticosterone was 7.6% compared to 0.4% in controls. Median % conversion of DOC to aldosterone was 6.2% compared to 0.06% in controls. Corticosteroids mediate a number of functions of mammalian brain, including blood pressure homeostasis, salt appetite and neuronal excitability. Local production of these steroids could have significant effects on these processes.

Universal TA cloning.

TA cloning is one of the simplest and most efficient methods for the cloning of PCR products. The procedure exploits the terminal transferase activity of certain thermophilic DNA polymerases, including Thermus aquaticus (Taq) polymerase. Taq polymerase has non-template dependent activity which preferentially adds a single adenosine to the 3'-ends of a double stranded DNA molecule, and thus most of the molecules PCR amplified by Taq polymerase possess single 3'-A overhangs. The use of a linearized "T-vector" which has single 3'-T overhangs on both ends allows direct, high-efficiency cloning of PCR products, facilitated by complementarity between the PCR product 3'-A overhangs and vector 3'-T overhangs. The TA cloning method can be easily modified so that the same T-vector can be used to clone any double-stranded DNA fragment, including PCR products amplified by any DNA polymerase, as well as all blunt- and sticky-ended DNA species. This technique is especially useful when compatible restriction sites are not available for the subcloning of DNA fragments from one vector to another. Directional cloning is made possible by appropriate hemi-phosphorylation of both the T-vectors and the inserts. With a single T-vector at hand, any DNA fragment can be cloned without compromising the cloning efficiency. The universal TA cloning method is thus both convenient and labor-saving.