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Biomedical subjects

C E Gomez-Sanchez

Publications and source records attributed to C E Gomez-Sanchez.

At least 19 recordsLinked to original sources
PubMed2000

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.

Animals↗
PubMed2000

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.

Alternative Splicing↗
PubMed2000

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.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗
PubMed2000

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.

Adrenal Glands↗
PubMed2000

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.

Aldosterone↗
PubMed1999

Metabolism of progesterone to DOC, corticosterone and 18OHDOC in cultured human melanoma cells.

We are now showing that cultured human melanoma cells can synthesize steroids such as corticosterone from progesterone or deoxycorticosterone. Corticosterone production is strongly responsive to deoxycorticosterone substrate addition (12-fold increase), but unresponsive to the adrenal stimulating factors ACTH and angiotensin II. This is the first demonstration that skin cells (malignant melanocytes) have the capability to synthesize 11-deoxycorticosterone, corticosterone, and 18-hydroxydeoxycorticosterone.

Adrenal Glands↗
PubMed1999

Maternal hypertension and progeny blood pressure: role of aldosterone and 11beta-HSD.

Epidemiological and experimental evidence suggests that gestational events modulate the level of blood pressure that will be "normal" for the individual as an adult. Glucocorticoid excess during gestation is associated with low birth weight, a large placenta, and adult hypertension in humans and animals. It has been proposed that the deficiency in placental 11beta-hydroxysteroid dehydrogenase activity in humans produces a gestational hormonal milieu, notwithstanding normal circulating levels of glucocorticoids, that predisposes the adult progeny to hypertension. Animal studies indicate that maternal hypertension, excess glucocorticoids, and hydroxysteroid dehydrogenase inhibition program adult blood pressure. Blood pressures of Sprague-Dawley rat dams were manipulated during gestation with continuous intracerebroventricular infusions of vehicle, aldosterone, 11alpha-hydroxyprogesterone, or carbenoxolone at doses known to produce hypertension with no renal effects or with subcutaneous infusions of larger, equally hypertensinogenic doses that produce systemic effects. Blood pressures of all treated dams were significantly greater (P<0.01) during gestation than those of the vehicle ICV control rats but not significantly different from each other. The blood pressures of both male and female progeny (n>/=6 per group, comprising representatives from at least 4 litters) were measured after 6 weeks of age. No significant difference was found in the blood pressure of the pups regardless of the maternal gestational blood pressure or treatment with an enzyme inhibitor, even after high-salt diet challenge.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1999

Levels of mineralocorticoids in whites and blacks.

Blacks appear, on average, to retain more Na than whites. A higher production rate of mineralocorticoids could explain the greater Na retention in blacks. Although production of aldosterone has been shown to be lower in blacks, the level of another mineralocorticoid may be increased. Plasma levels of deoxycorticosterone and cortisol were measured in young whites (n=23; age=16.4+/-3.1[SD] years) and young blacks (n=25; age=13.8+/-1.3 years). Blacks had lower plasma levels of renin activity and aldosterone and lower urinary aldosterone excretion rates; thus, they appeared to be representative of blacks that retain additional Na. Plasma deoxycorticosterone levels were lower in blacks than in whites both at baseline (247+/-161 versus 381+/-270 pmol/L, P=0.048) and after stimulation with adrenocorticotropic hormone (822+/-294 versus 1127+/-628 pmol/L at 30 minutes, P=0.047; 925+/-366 versus 1440+/-834 pmol/L at 60 minutes, P=0.013). Cortisol levels were also lower in blacks at baseline (P=0.014) but were not significantly different from levels in whites after stimulation with adrenocorticotropic hormone. In a larger cohort of 407 whites (age=12.0+/-2.9 years) and 247 blacks (age=12.9+/-3.1 years), 18-hydroxycortisol excretion rates were also lower in blacks (P=0. 021). In conclusion, increased Na retention in blacks does not appear to be secondary to increased production of either aldosterone, deoxycorticosterone, cortisol, or 18-hydroxycortisol. A primary renal mechanism may mediate the increase in Na reabsorption in blacks.

Adolescent↗
PubMed1998

Impaired neuroendocrine response mediates refractoriness to cardiopulmonary resuscitation in spinal anesthesia.

OBJECTIVE: To determine the extent of neurogenic control on adrenal secretion in a canine model of high spinal anesthesia and cardiac arrest. DESIGN: Randomized, controlled, acute intensive study. SETTING: University intensive care laboratory. SUBJECTS: Nineteen healthy, anesthetized, mongrel dogs. INTERVENTIONS: Cardiac arrest was induced in 11 spinally anesthetized dogs and 8 sham-control animals; cardiopulmonary resuscitation (CPR) was started 60 secs later. Epinephrine was injected at 4 mins and every 2 mins thereafter. Arterial blood samples were obtained before anesthesia, before arrest, and after 1, 3, 5, 7, 9, and 11 mins of CPR. MEASUREMENTS AND MAIN RESULTS: At 1 and 3 mins after cardiac arrest, the control group exhibited significant increases of epinephrine and norepinephrine concentrations (p < .05) that were absent in the spinal anesthesia group. Plasma renin increased in both groups whereas aldosterone and cortisol remained unchanged. CONCLUSIONS: Spinal anesthesia abolishes the catecholamine release that follows cardiac arrest, while a previously postulated direct adrenal effect of hypoxia stimulating catecholamine release was not confirmed in these experiments. Since epinephrine treatment restores coronary perfusion pressure (CPP) during CPR, we conclude that catecholamine deficiency is the most likely mechanism for inadequate CPP during CPR conducted in the presence of spinal anesthesia.

Adrenal Glands↗
PubMed1998

Development of adrenal zonation in fetal rats defined by expression of aldosterone synthase and 11beta-hydroxylase.

The adult rat adrenal cortex is comprised of three concentric steroidogenic zones that are morphologically and functionally distinguishable: the zona glomerulosa, zona intermedia, and the zona fasciculata/reticularis. Expression of the zone-specific steroidogenic enzymes, cytochrome P450 aldosterone synthase (P450aldo), and P450 11beta hydroxylase (P45011beta), produced by the zona glomerulosa and zona fasciculata/reticularis, respectively, can be used to define the adrenal cortical cell phenotype of these two zones. In this study, immunohistochemistry and in situ hybridization were used to determine the ontogeny of expression of P450aldo and P45011beta to monitor the pattern of development of the rat adrenal cortex. RIA was used to measure adrenal content of aldosterone and corticosterone, the resulting products of the two enzymatic pathways. Double immunofluorescent staining for both enzymes at gestational day 16 (E16) showed P45011beta protein expressed in cells distributed throughout most of the adrenal intermixed with a separate, but smaller, population of cells expressing P450aldo protein. Whereas expression of P45011beta protein retained a similar pattern of distribution from E16 to adulthood (ignoring distribution of SA-1 positive, presumptive medullary cells), P450aldo protein changed its pattern of distribution by E19, becoming localized in a discontinuous ring of cells adjacent to the capsule. By postnatal day 1, P450aldo protein distribution was similar to that observed in adult glands; P450aldo-positive cells formed a continuous zone underlying the capsule. In situ hybridization showed that the pattern of P45011beta messenger RNA expression paralleled protein expression at all times, whereas P450aldo messenger RNA paralleled protein at E19 and after, but was undetectable before E19. However, adrenal aldosterone and corticosterone, as measured by RIA, were detected by E16, supporting the functional capacity of both phenotypes for all ages studied. These data suggest that the development of the adrenal zona glomerulosa occurs in two distinct phases; initial expression of the glomerulosa phenotype in scattered cells of the inner cortex before E17, followed by a change in distribution to the outer cortex between E17 and E19. It is hypothesized that this change in distribution occurs via cell differentiation, rather than cell migration, and that a possible regulator of these events is the fetal renin-angiotensin system.

Adrenal Glands↗
PubMed1997

Regulation of the 11 beta-hydroxysteroid dehydrogenase in the rat adrenal. Decrease enzymatic activity induced by ACTH.

Patients with ectopic ACTH syndrome often develop hypertension and hypokalemic alkalosis with an abnormal increase in the ratio of plasma cortisol to cortisone, indicating that 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) activity is inhibited. Inhibition of 11 beta HSD allows access of cortisol or corticosterone to the mineralocorticoid receptor where it act as a mineralocorticoid. Two isozymes, 11 beta HSD-1 and 11 beta HSD-2, have been cloned and characterized. The rat adrenal expresses the mRNAs for 11 beta HSD-2 and, in lesser amounts, 11 beta HSD-1. We investigated the effect of ACTH on the 11 11 beta HSD-2 activity in the rat adrenal. Rat adrenal cells zone fasciculata (ZF) were dispersed and incubated separately with increasing concentrations of ACTH for 90 min, and secretion of corticosterone (B) and 11-dehydrocorticosterone (A) in the media was measured by enzyme-linked immunoabsorbent assays (ELISA). The conversion of [3H]B to [3H]A in the presence of 0.5 mM NAD+ was evaluated in microsomes prepared from dispersed cells preincubated for 30 min with cyanoketone and metyrapone followed by incubation for 30 min with the same inhibitors, with and without 10 nM ACTH. The dispersed cells of the ZF produced significant amounts of A which increased with ACTH. The basal B/A ratio was 0.97 +/- 0.05. ACTH caused a concentration-dependent increase in the ratio of B/A with a maximum ratio of 9.58 +/- 0.20. ACTH also inhibited the conversion of [3H]B to [3H]A in microsomes in which endogenous B production was inhibited by cyanoketone and metyrapone. ACTH did not change the K(m) for B conversion, but the Vmax was reduced significantly (1.73 +/- 0.43 pmol/min. mg protein), indicating that ACTH suppressed the 11 beta HSD-2 in a noncompetitive fashion. Dibutyryl cyclic AMP (dcAMP) also produced a concentration-dependent increase in the B/A ratio, but various concentrations of calcium did not affect the enzyme activity. In summary, adrenal cells treated with ACTH results in a significant increase in the ratio of B/A in the ZF owing a noncompetitive inhibition of the 11 beta HSD-2 via the ACTH receptor.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1997

The sheep kidney contains a novel unidirectional, high affinity NADP(+)-dependent 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD-3).

The 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) enzymes convert corticosterone and cortisol to 11-dehydrocorticosterone and cortisone, and are thought to convey extrinsic specificity to the mineralocorticoid receptor by limiting access of the relatively more abundant glucocorticoids to it. Two different 11 beta-hydroxysteroid dehydrogenases (11 beta-HSD) have been described and cloned. The liver-type, NADP(+)-dependent 11 beta-HSD-1, has an affinity in the micromolar range and bidirectional activity. The NAD(+)-dependent 11 beta-HSD-2 has a higher affinity, in the nanomolar range, and exhibits only oxidase activity. 11 beta-HSD-2, because of its affinity and co-localization with the mineralocorticoid receptor, is likely to serve as the "gatekeeper" for the mineralocorticoid receptor in the kidney. Although the rat kidney expresses both isoforms, only the high-affinity, NAD(+)-dependent 11 beta-HSD-2 has been reported in the sheep kidney. We found both 11 beta-HSD NAD(+)- and NADP(+)-dependent activities in sheep kidney to be present. The NAD(+)-dependent activity exhibited a Km similar to that reported in the literature, 3.85 +/- 1.28 nM for corticosterone and 21.3 +/- 5.8 for cortisol, was distributed in approximately equal amounts between microsomes and nuclei, and was unidirectional, converting corticosterone to 11-dehydrocorticosterone. The enzyme exhibited prominent substrate inhibition. The NADP(+)-dependent activity had a Km for corticosterone of 4 +/- 1.3 nM for a Km for cortisol of 35.2 +/- 2 nM, 100-fold lower than that described for the 11 beta-HSD-1 in the liver of sheep and other species, and was more prevalent in the microsomes than the nuclei. This enzyme was not inhibited by its substrate. The NAD(+)-dependent activity was approximately 3-10 times greater than the NADP(+)-dependent activity when incubated with 5 nM corticosterone substrate, but had similar activity when incubated with 100 nM substrate concentrations. CHOP cells (a modified Chinese hamster ovary cell line) transiently transfected with the sheep 11 beta-HSD-2 plasmid exhibited a marked preference for NAD+ as co-factor. Oxidation of corticosterone by transfected cells in the presence of NADP+ was present, but minimal; NADP+ did not support the metabolism of cortisol, the primary glucocorticoid of sheep. These data suggest the existence of another NADP(+)-dependent enzyme, 11 beta-HSD-3, which, because of its high affinity and unidirectional oxidase activity, may play a physiological role in the modulation of glucocorticoid binding to both the mineralocorticoid and glucocorticoid receptors.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1997

Inhibition of steroidogenesis in rat adrenal cells by 18-ethynyldeoxycorticosterone: evidence for an alternative pathway of aldosterone biosynthesis.

The effect of the mechanism-based inhibitor 18-ethynyldeoxycorticosterone (18-E-DOC) on the late steps of the aldosterone biosynthetic pathway was examined in freshly isolated cells of the zona glomerulosa (ZG) and fasciculata (ZF) from rat adrenal glands. ZG synthesis of aldosterone was inhibited by 18-E-DOC in a time- and concentration-dependent manner with a Ki of approximately 0.05 microM. The maximal degree of inhibition of ZG production of aldosterone and 18-hydroxycorticosterone (18-OH-B) was approximately 80%. ZF cells, perhaps surprisingly, were found to secrete 18-OH-B at levels approximately one-third to one-fourth those of ZG cells and the Ki of 18-E-DOC inhibition of 18-OH-B secretion was approximately 10 microM for ZF cells, 200-fold higher than for ZG cells. The inhibitor had no effect on the secretion of corticosterone by either ZG or ZF, and the secretion of 18-hydroxydeoxycorticosterone (18-OH-DOC) by both the ZG and ZF was inhibited only to a minor degree. 18-E-DOC inhibited the biosynthesis of aldosterone by ZG cells incubated with 10 microM added DOC or 18-OH-DOC by approximately 75%, similar to the degree of inhibition of aldosterone biosynthesis from endogenous substrate, whereas ZF biosynthesis of 18-OH-B from either substrate was inhibited by less than 40%. ZF cells do not express aldosterone synthase, the only enzyme known to convert 18-OH-DOC into 18-OH-B. Incubation of MA-10 cells stably transfected with the cDNA of the rat aldosterone synthase with 18-E-DOC resulted in a complete inhibition of the conversion of DOC to aldosterone with a Ki of approximately 0.02 microM. In addition, transfected cells expressing 11beta-hydroxylase convert DOC to 18-OH-B in very small quantities only and cannot convert 18-OH-DOC to 18-OH-B. These data suggest that neither 11beta-hydroxylase nor aldosterone synthase are responsible for the biosynthesis of 18-OH-B by ZF cells from DOC or 18-OH-DOC, that 20% of aldosterone synthesis appears not to be attributable to the actions of aldosterone synthase and that an unknown CYP11B enzyme is also involved in the biosynthesis of 18-OH-B.

Adrenal Glands↗
PubMed1997

Cloning of two alternatively spliced 21-hydroxylase CDNAs from rat adrenal.

Interest in extra-adrenal corticosteroid synthesis has been revived by technological advances and the quest for answers to clinical problems. The cytochrome P450 21-hydroxylase converts progesterone to deoxycorticosterone, the obligatory substrate for the production of the main adrenal steroids aldosterone, cortisol and corticosterone. The rat P450 21-hydroxylase was cloned and two constructs, 21OH-5 and 21OH-6, sequenced. The constructs are similar, except that 21OH-6 has three additional major insertions of 64, 70 and 84 bp, a 3 bp deletion, and four extra base pairs immediately before the poly-A sequence. The entire coding region of 21OH-5 has 87 and 71% homology with the mouse and human 21-hydroxylase cDNA, respectively, whereas the encoded protein has 84 and 65% homology. Reverse transcriptase-polymerase chain reaction (RT-PCR) combined with Southern blot demonstrated expression of both transcripts in the kidney, aorta, liver, cerebellum, hypothalamus and brain stem, heart and cerebrum, but not the hippocampus, in addition to the adrenal. The entire coding region of 21OH-5 and the corresponding region of 21OH-6 including the three introns were cloned into pCR3 and the plasmids transiently transfected into COS-7 cells. Only 21OH-5 was translated into active protein, converting approximately 64% of 3H-progesterone to deoxycorticosterone in 2 h.

Alternative Splicing↗
PubMed1997

Analysis of phenotypic consequences of renin gene polymorphism in Lyon rats.

OBJECTIVE: To investigate phenotypic consequences of renin gene polymorphism between Lyon hypertensive (LH) and normotensive (LN) rats because previously we demonstrated cosegregation of the LH allele with increased blood pressure in a cross of LH with LN rats. DESIGN: Two studies were conducted. Study 1 used a cohort of male F2 rats from a LH x LN cross. Eighty-two rats homozygous for the hypertensive (HH) renin gene allele were compared with 82 rats homozygous for the normotensive (NN) allele. Urinary steroid excretion was measured in 24 h urine samples collected from rats aged 6 weeks. The direct aortic blood pressure was recorded in 30-week-old rats and, after they had been killed, their kidney renin concentration (KRC) was measured. In study 2, renin, angiotensinogen and angiotensin converting enzyme plasma concentrations and renin messenger RNA (mRNA) levels were measured in renal and extra-renal tissues from 6- and 25-week-old LH and LN parental and HH and NN F2 male rats. METHODS: Urinary steroids and plasma components of the renin-angiotensin system (RAS) were measured using specific radioimmunoassays. mRNA levels were quantified by northern blotting. RESULTS: In study 1, HH F2 rats had a higher blood pressure (151.5 +/- 8.2 versus 146.0 +/- 7.4 mmHg, P < 0.001) and a lower KRC (514 +/- 203 versus 666 +/- 304 micrograms A1/h per g cortex, P < 0.01) than did NN rats aged 30 weeks. In covariate analysis the decrease in KRC in HH rats was attributable to their increased blood pressure rather than to the renin genotype. The renin genotype of rats aged 6 weeks was not associated with a change in the urinary excretion of aldosterone, desoxycorticosterone, corticosterone or 18-hydroxy desoxycorticosterone. In study 2, we found no difference either in plasma levels of RAS components or in renal or extrarenal renin mRNA levels either between parental LH and LN rats or between HH and NN F2 rats apart from a higher plasma renin concentration in LH rats aged 6 weeks. Renal, but not extra-renal, renin mRNA levels declined with age. CONCLUSIONS: We found no evidence of a renin genotype-dependent phenotypic difference in the RAS that could account for the effect of the renin locus on blood pressure in Lyon rats. Our findings suggest that the effect of the locus on blood pressure might be due to an as yet unidentified gene linked to renin.

Animals↗
PubMed1997

Aldosterone biosynthesis in the rat brain.

Messenger RNA (mRNA) for enzymes involved in adrenal steroid biosynthesis are expressed in the brain, and the coded enzymes have been shown to be active. The expression of mRNA for the cytochrome P-450 enzyme aldosterone synthase, crucial for the final step in the synthesis of aldosterone and the synthesis of aldosterone was studied in several anatomic areas of the rat brain. Expression of the mRNA for the aldosterone synthase was demonstrated by RT-PCR/Southern blot in adrenal, aorta, hypothalamus, hippocampus, amygdala, cerebrum, and cerebellum. Incubation of brain minces from intact and adrenalectomized rats demonstrated the synthesis of corticosterone and aldosterone from endogenous precursors. Incubations of brain minces with [1,2(3)H]-deoxycorticosterone, followed by extraction and three different successive TLCs, demonstrated the presence of labeled aldosterone, corticosterone, and 18-hydroxy-deoxycorticosterone. Incubation, in the presence of 10 microM cortisol or metyrapone, inhibited the synthesis of aldosterone or both aldosterone and corticosterone, respectively. These studies indicate that the rat brain has the enzymatic machinery for the synthesis of adrenal corticosteroids and is capable of synthesizing aldosterone. Aldosterone synthesized in the brain might play a paracrine role in the regulation of blood pressure.

Aldosterone↗
PubMed1996

11 beta-hydroxysteroid dehydrogenases of the choriocarcinoma cell line JEG-3 and their inhibition by glycyrrhetinic acid and other natural substances.

Mineralocorticoid receptor (MR) selectivity for aldosterone is thought to be exerted by enzymes which inactivate competing glucocorticoids before they bind the receptor. Two different 11 beta-hydroxysteroid dehydrogenases (11 beta-HSD) have been described. 11 beta-HSD-1 is NADP(+)-dependent and has a Km in the micromolar range and bidirectional activity. 11 beta-HSD-2 is NAD(+)-dependent, has a Km in the nanomolar range, exhibits only oxidase activity, and colocalizes with the MR in the kidney, so is likely to serve as the gatekeeper for the MR. We have further characterized 11 beta-HSD activity in JEG-3 cells, a cell line derived from a human choriocarcinoma which was reported to have only the high affinity, NAD(+)-dependent 11 beta-HSD-2. We found that the Km for the conversion of corticosterone to 11-dehydrocorticosterone in intact cells and homogenates was about 16 nM. NAD(+)-dependent corticosterone conversion was equal in the nuclear and mitochondrial fractions and less, but significant, in the microsomal fraction. A high affinity, Km = 40 nM, NADP(+)-dependent enzyme was also found in homogenates. The subcellular distribution of this high affinity activity was greatest in the mitochondria, less in the nuclei, and even less, but still significant, in microsomes. Because of its cofactor dependency, high affinity, and different subcellular distribution, we suggest that this enzyme is neither the 11 beta-HSD-1 nor the 11 beta-HSD-2 and have named it 11 beta-HSD-3. Conversion of 11-dehydrocorticosterone to corticosterone did not occur in intact cells or in homogenates incubated with NADH or NADPH. Enzyme activity in intact cells was inhibited by glycyrrhetinic acid, carbenoxolone, progesterone, 5 beta-dihydroprogesterone, and 5 alpha-dihydroprogesterone, but not bile acids.

11-beta-Hydroxysteroid Dehydrogenases↗