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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
PubMed2001

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.

Adrenal Cortex↗
PubMed2001

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.

Blood Pressure↗
PubMed2001

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.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗
PubMed2001

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.

Aldosterone↗
PubMed2001

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.

Adrenal Glands↗
PubMed2001

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.

Adrenal Cortex↗
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↗
PubMed2000

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.

Cloning, Molecular↗
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↗