PubMed Health⌕ Search

Biomedical subjects

J G LeHoux

Publications and source records attributed to J G LeHoux.

At least 19 recordsLinked to original sources
PubMed2000

Control of CYP11B2 gene expression through differential regulation of its promoter by atypical and conventional protein kinase C isoforms.

We reported previously that the protein kinase C (PKC) inhibitor GF109203X stimulated the hamster CYP11B2 promoter activity in transfected NCI-H295 cells. PKCalpha, -epsilon, and -zeta were detected in hamster adrenal zona glomerulosa and NCI-H295 cells, and PKCtheta in NCI-H295 cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA) inhibited basal and stimulated cytochrome P450 aldosterone synthase mRNA expression by angiotensin (AII), dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP), or KCl in NCI-H295 cells. Basal CYP11B2 promoter activity was inhibited in cells cotransfected with constitutively active (CA) PKCalpha, -epsilon, and -theta mutants, whereas it was increased with CA-PKCzeta. Dominant negative (DN) PKCalpha, -theta, -epsilon, and -zeta mutants stimulated the promoter activity. AII-, KCl-, and Bt2cAMP-stimulatory effects were abolished in cells cotransfected with CA-PKCalpha, -epsilon, or -theta. The effect of Bt2cAMP was abolished by CA-PKCzeta but AII and KCl were still able to enhance the promoter activity. DN-PKCalpha, -epsilon, -theta, or -zeta did not inhibit these effects. Gö6976 enhanced promoter activity, providing further evidence that PKCalpha was involved. Various CYP11B2 promoter constructs were used to identify the area associated with TPA and PKC inhibition. TPA and CA-PKCalpha, -epsilon, or -theta abolished the effects of AII, KCl, and Bt2cAMP on the activity of -102 and longer constructs. In summary, our findings suggest that the hamster CYP11B2 gene is under differential control by conventional (alpha) and atypical (zeta) PKC.

Animals↗
PubMed2000

Molecular modeling of the hamster adrenal P450C17.

The cytochrome P450C17 (C17) is the steroidogenic enzyme responsible for the conversion of pregnenolone and progesterone to dehydroepiandrosterone (DHEA) and delta4-androstenedione (AD) respectively. This conversion is achieved by two enzymatic activities, 17alpha-hydroxylase and 17,20-lyase, located at the same active site. In man, the adrenal C17 basically only produces DHEA. We have shown that the hamster adrenal C17 produces DHEA as well as AD. Moreover, the hamster like man produces cortisol as its major glucocorticoid. We can thus compare the hamster and human adrenal C17, and use their differences in order to elaborate a strategy for structure-function studies. We have thus engineered hamster adrenal C17 mutants which possess modified enzymatic activities. We also proceeded to elaborate a three-dimensional model of the hamster C17 to visualise the structural impact of these mutations. This model demonstrates that the mutations created are not localised at the active site, but rather in surrounding regions. These could affect the conformation of the active site, in turn, modulating the 17alpha-hydroxylase and 17,20-lyase activities. For example, the mutation T202N is located next to Val 482 and Val 483 which compose the roof of the active site. This mutation decreased both 17alpha-hydroxylase and 17,20-lyase activities, indicating the importance of the roof of the active site for general functionality of the C17.

Adrenal Glands↗
PubMed2000

Regulation of CYP11B2 gene expression by protein kinase C.

Bisindolylmaleimide, a protein kinase C (PKC) inhibitor, was shown to stimulate the hamster CYP11B2 promoter activity in transfected NCI-H295 cells. In this study we have found that TPA, an activator of PKC, also inhibited the hamster CYP11B2 promoter activity. DAG-dependent PKC alpha and PKC epsilon, and atypical PKC zeta were detected in hamster adrenal zona glomerulosa, whereas the isoforms alpha, epsilon, zeta and theta were found in NCI-H295 cells. CYP11B2 promoter activity was inhibited in cells co-transfected with constitutively active PKC alpha and epsilon mutants, whereas it was increased with the constitutively active PKC zeta mutant. Dominant negative PKC alpha, epsilon and zeta mutants stimulated the promoter activity. Gö6976, a specific inhibitor of classical PKCs, enhanced promoter activity, providing further evidence that PKC alpha, the only classical PKC revealed in hamster adrenal and NCI-H295 cells, was involved in the promoter inhibition.

Adrenal Glands↗
PubMed2000

Age-related decrease of dehydroepiandrosterone concentrations in low density lipoproteins and its role in the susceptibility of low density lipoproteins to lipid peroxidation.

The incidence of atherosclerosis and related diseases increases with age. The aging process may enhance lipoprotein modification, which leads to an increase in the susceptibility of low density lipoprotein (LDL) and high density lipoprotein (HDL) to oxidation. Dehydroepiandrosterone (DHEA), the most abundant steroid hormone in humans, has been shown to have antiatherogenic effects. This hormone also decreases dramatically with age. In the present study, we were interested in determining the presence of DHEA/DHEAS (dehydroepiandrosterone sulfate) and changes in their concentrations in HDL and LDL lipoproteins with age. Moreover, we studied the susceptibility of LDL to oxidation with age in the presence or absence of vitamin E or DHEA. We demonstrated that vitamin E is unable to restore the decreased resistance to oxidation of LDL from elderly subjects to that of LDL obtained from young subjects. Furthermore, our results provide evidence that DHEA is an integral part of LDL and HDL and disappears to almost nondetectable levels during aging. The DHEA incorporated into the LDL from elderly subjects increased LDL resistance to oxidation in a concentration-dependent manner. The increased resistance provided by DHEA was higher than that with vitamin E. DHEA seems to act either by protecting vitamin E from disappearance from LDL under oxidation or by scavenging directly the free radicals produced during the oxidative process. Our results suggests that DHEA exerts an antioxidative effect on LDL, which could have antiatherogenic consequences. Careful clinical trials of DHEA replacement should determine whether this ex vivo effect could be translated into any measurable antiatherogenic (cardioprotective) action.

Adult↗
PubMed1999

Mutations in the gene encoding the alpha-subunit of the Gs protein in molar pregnancy.

Molar pregnancy is a gestational trophoblastic disease associated with a trophoblastic proliferation and a protein synthesis alteration. It is characterized by the presence of hydatiform moles, which are fluid-filled cysts derived from the chorionic villi of the placenta. Recent studies have reported a reduced expression of several types of G proteins including Gsalpha in molar pregnancies suggesting alterations in G protein structure in hydatiform moles. To identify mutations that lead to Gsalpha deficiency, we isolated genomic DNA from hydatiform moles and used polymerase chain reaction to amplify all exons of the Gsalpha gene. Amplified Gsalpha gene fragments were analyzed by sequencing using the dideoxy chain termination method. Tissues obtained from three complete hydatiform moles and one partial hydatiform mole were examined. We have identified a heterozygous 8-bp deletion in exon 10 of the Gsalpha gene, in two complete hydatiform moles, that had evidence for a dysfunctional Gsalpha protein. This deletion produced a truncated protein. We have also identified a heterozygous polymorphism in exon 5 in two complete hydatiform moles, and a homozygous substitution (A-->G) in intron 5 of the Gsalpha gene in the other complete hydatiform mole; these two last types of mutations should not have any effects on protein activity.

Adult↗
PubMed1998

Inhibitory effects of Serenoa repens on the kinetic of pig prostatic microsomal 5alpha-reductase activity.

The pathogenesis of benign prostatic hyperplasia is linked to the accumulation of dihydrotestosterone (DHT), the active form of testosterone (T), in prostatic tissue. We have defined characteristics of 5alpha-reductase enzyme which catalyzes the conversion of T into DHT in prostatic microsomes of growing pigs. Peaks for the 5alpha-reductase activity were found at pH 5.5 and 8.0, which indicates the presence of both type 1 and type 2 isozymes. Kinetic parameters of porcine 5alpha-reductase in the presence of Serenoa repens extracts revealed uncompetitive, noncompetitive, and mixed types of inhibitions. Our results show the inhibitory action of S. repens on prostate porcine microsomal 5alpha-reductase activity.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1998

Characterization of cDNAs encoding isoforms of hamster 3 beta-hydroxysteroid dehydrogenase/delta 5-->4 isomerase.

Western blot analyses of various hamster tissues reveal high levels of expression of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in adrenal and liver, and moderate levels of expression in kidney. The expression in liver is sexually dimorphic; very high levels of protein are observed in adult male liver but very low levels are seen in the female liver. Three distinct cDNAs encoding isoforms of 3 beta-HSD were isolated from hamster cDNA libraries. The type 1 isoform is a high-affinity dehydrogenase/isomerase expressed in adrenal and male kidney. The type 2 isoform is also a high-affinity dehydrogenase/isomerase expressed in kidney and male liver. The type 3 enzyme is a 3-ketosteroid reductase expressed predominantly in kidney. Sequencing of the clones showed that all three are structurally very similar, although types 1 and 2 share the greatest degree of similarity. Immunohistochemical staining for 3 beta-HSD in the adrenal was found throughout the adrenal cortex. In the kidney staining was confined to tubules, and in the liver, heavy staining was found in hepatocytes. The cloning of cDNAs for 3 beta-HSD from the liver and kidney should help in elucidating the function of this enzyme in these tissues.

Adrenal Glands↗
PubMed1998

Transcriptional activity of the hamster CYP11B2 promoter in NCI-H295 cells stimulated by angiotensin II, potassium, forskolin and bisindolylmaleimide.

We studied the regulation of the hamster CYP11B2 gene in the NCI-H295 cell line, which is known to produce aldosterone in response to stimulation by angiotensin II (AII) and KCl. Ten deletion plasmids harboring the 5'-untranslated region of the CYP11B2 gene were used for chloramphenicol acetyltransferase (CAT) assays. Transient transfections showed progressively increasing basal promoter activity by constructs beyond the TATA box, with a peak occurring with the -167 bp construct which contains putative Adl, Ad2, Ad5 and the newly reported -143/-161 cis-element sequences. The promoter activity was lower with the construct containing the putative Ad3 cis-element and increased with longer constructs. This indicates the presence of both inhibitory and stimulatory cis-elements in this area of the gene. Expression of the reporter gene of all constructs was stimulated by AII and KCl, with the exception of the construct containing only the TATA box, which showed 6-fold and 10-fold increases occurring with the -167 bp deletion plasmid. The patterns of increase in CAT activity with AII and KCI treatment were similar, showing that these two regulators can stimulate hamster CYP11B2 promoter activity through common cis-elements. The calcium channel antagonist nifedipine blocked the stimulatory effects of KCl on CAT activity, showing the involvement of calcium channels in the regulation of CYP11B2 gene transcription by KCl. 12-O-Tetradecanoylphorbol 13-acetate, a known stimulator of the protein kinase C (PKC) signaling pathway, was without significant effect on CAT activity. Bisindolylmaleimide, a specific inhibitor of PKC, had a significant enhancing effect (3.4- to 6-fold), indicating that PKC may negatively regulate the expression of the hamster CYP11B2 gene in NCI-H295 cells. A mutation was induced in the sequence -143/-161 of the - 350 bp construct in order to determine its importance in the regulation of hamster CYP11B2 promoter activity. The stimulatory effects of AII, KCl, forskolin and bisindolylmaleimide on CAT activity were significantly less in the mutant than in the wild type. These results confirm that this cis-element is necessary in maintaining a high level of transcriptional activity in stimulated NCI-295H cells. In conclusion, using NCI-295H transfected cells, we have found that the 5'-untranslated region of the hamster CYP11B2 gene possesses transcriptional activity with stimulatory and also inhibitory cis-elements; CYP11B2 promoter activity can be stimulated by AII, KCl, forskolin, dibutyryl cAMP and bisindolylmaleimide. Our results suggest that this gene is positively regulated through the protein kinase A signaling pathway and through calcium channels, whereas PKC may have a negative regulatory effect upon the transcription of the CYP11B2 gene. Furthermore, we have shown that the cis-element -143/-161 in the 5'-untranslated region of the hamster CYP11B2 gene is important in maintaining a high level of promoter activity in stimulated NCI-295H cells.

Aldosterone↗
PubMed1998

In vivo effects of adrenocorticotrophin on the expression of the hamster steroidogenic acute regulatory protein.

In this study, we report the cDNA cloning of hamster adrenal steroidogenic acute regulatory (StAR) protein and the effect of adrenocorticotrophin (ACTH) on its expression in vivo. A hamster adrenal cDNA library was screened using an 852 bp fragment obtained by polymerase chain reaction; this fragment corresponds to the entire coding sequence (CDS) of the hamster adrenal StAR cDNA. Ten clones of different lengths were isolated and sequenced. The longest clone was 1564 bp and contained 34 bp in the 5'-untranslated region, 852 bp in the CDS, and 678 bp in the 3'-untranslated region (3'-UTR). Two polyadenylation signal sequences were found in the 3'-UTR. The CDS of the ten isolated clones was identical, but six of these lacked the last 132 nucleotides in the 3'-UTR, thus indicating that they had used the first polyadenylation signal. The hamster StAR protein contains 284 amino acid residues, and is 91.9% homologous to mouse, 90.5% to rat, 86.4% to human, 85% to porcine, and 82.5% to bovine StAR protein. Southern blot analysis indicated the presence of only one StAR gene in the hamster genome. Northern blotting analysis revealed the presence of the StAR mRNA in male and female steroidogenic tissues, namely adrenals and gonads, but not in the liver or in the kidneys of either sex. Three mRNA species of 1.7, 3.1 and 5.3 kb were found in whole hamster adrenals. Administration of ACTH to hamsters provoked increases (two- to threefold) in the adrenal content of the StAR mRNA within 1 h in vivo. Western blotting analysis on adrenal mitochondria showed that the level of StAR protein was also significantly elevated (1.5-fold) 1 h after ACTH treatment.

Adrenal Glands↗
PubMed1997

Immunolocalization and biochemical determination of cytochrome P450C17 in adrenals of hamsters treated with ACTH.

We used an anti-rat adrenal cytochrome P450C17 (P450C17) antibody to perform immunofluorescence and also immunogold electron microscopic studies to determine the zonal and intracellular distribution of P450C17 in hamster adrenals. Because P450C17 activity is regulated mainly by adrenocorticotropin (ACTH), its zonal and intracellular localization was also analyzed after ACTH treatment. The effect of ACTH treatment on protein concentration was also investigated by Western blotting analysis. By immunofluorescence, we found P450C17 to be confined to the zona fasciculata (ZF) in the hamster, in contrast to other small rodents, which do not express P450C17 in their adrenals. After treatment with ACTH, the thickness of the ZF remained unchanged compared to that of control animals, whereas a marked increase in fluorescence intensity was observed. In addition, dispersed cells in the zona reticularis (ZR) showed positive staining after ACTH treatment. Immunocytochemistry with colloidal gold showed P450C17 to be localized and importantly increased only in the cytoplasmic areas between the mitochondria of ZF cells of ACTH-treated animals. These areas are predominantly occupied by elements of the endoplasmic reticulum and other unidentified organelles. Immunoblotting analysis of whole glands revealed a single protein band at approximately 55 kD, which reacted with the 450C17 antibody. After stimulation with ACTH injected at 5-hr intervals over a period of 20 hr, P450C17 protein concentrations were considerably greater than in control animals. In conclusion, P450C17 is located not over mitochondria but probably in the endoplasmic reticulum of the ZF cells in hamster adrenals. Treatment with ACTH induced expression of cytochrome P450C17 in ZF cells, increasing its production in these cells without stimulating cell proliferation.

Adrenal Glands↗
PubMed1997

Alternate splicing of the RNA for hamster type 2 3 beta-hydroxysteroid dehydrogenase/delta 5-->4isomerase.

Complementary DNAs encoding the hamster type 2 3 beta-hydroxysteroid dehydrogenase/delta 5-->4 isomerase were isolated from liver and kidney cDNA libraries. Nine clones were isolated containing identical coding and 3' untranslated sequences. However, six of the clones contained a 68-nucleotide stretch in the 5' untranslated region that was missing in the other three clones. Primers were designed to flank this region and the polymerase chain reaction (PCR) was performed on hamster liver and adrenal RNA. Two PCR products were amplified of the predicted molecular sizes and with the expected sequence. Primers were then designed to amplify sequences encompassing this region from hamster genomic DNA. Sequencing of the resultant PCR products demonstrated that the 68-nucleotide stretch missing in some transcripts corresponded exactly to the second of three exons identified. We conclude that the 5' untranslated region of this mRNA is transcribed from at least three exons, and that the sequence of the second of these exons is spliced out of some of the RNA transcripts.

Alternative Splicing↗
PubMed1996

The hamster adrenal cytochrome P450C11 has equipotent 11beta-hydroxylase and 19-hydroxylase activities, but no aldosterone synthase activity.

We have isolated a hamster adrenal P45OC11 cDNA which shared 90 and 84% homology, respectively, with the nucleotide sequence and the amino acid sequence of the hamster adrenal P450aldo. Both P450C11 and P450aldo cDNA coding sequences were inserted in the plasmid pBluescript SK, transcribed and then translated using a rabbit reticulocyte system in the presence of [35S]methionine. The reaction products were immunoprecipitated with an anti-bovine P450C11 antibody for P450C11 and with an anti-hamster P450aldo for P450aldo. Immunoprecipitated proteins were analyzed by polyacrylamide gel electrophoresis. A single 35S-labeled protein band was detected for P450C11 and for P450aldo, respectively. P450C11 and P450aldo cDNAs were then both inserted into the expression vector pCMV5 containing a viral sequence specific for the attachment of ribosomes to mRNA. These constructions were transfected in COS-1 cells. 24 h after transfection, the presence of P450C11 and P450aldo mRNAs was determined by Northern blot analysis. In a time study experiment we found that P450C11 transformed the labeled-steroid into [14C]corticosterone, [14C]19-OH-deoxycorticosterone and [14C]18-OH-deoxycorticosterone in ratios of 1:1.11:0.07, after 2 h of incubation; no [14C]aldosterone could be detected. Cells transfected with plasmids harboring the P450aldo cDNA transformed [14C]deoxycorticosterone to [14C]corticosterone, [14C]aldosterone, [14C]18-OH-corticosterone, [14C]18-OH-deoxycorticosterone, [14C]19-OH-deoxycorticosterone and [14C]11-dehydrocorticosterone in ratios of 1:0.25:0.45:0.04:0.04:0.04 after 12 h of incubation. These results indicate that one P450 catalyzes the ultimate step of glucocorticoid formation and a separate P450 is involved in the final steps of aldosterone formation in hamster adrenals. The capacity of the hamster adrenal P450C11 to hydroxylate at positions 11beta and 19 in nearly equal ratio makes this animal an excellent model to study the mechanism of synthesis and inhibition of 19-OH-deoxycorticosterone, the precursor of 19-nor-deoxycorticosterone, a very potent mineralocorticoid involved in the development of essential hypertension.

Adrenal Glands↗
PubMed1996

Adrenocorticotropin regulates the level of the steroidogenic acute regulatory (StAR) protein mRNA in hamster adrenals.

In this study, we report the cloning of a StAR cDNA from a hamster adrenal cDNA library. The library was screened using a PCR fragment specific for the hamster adrenal StAR cDNA. Several clones of different lengths were obtained and one of these was sequenced. Northern blotting analysis revealed the presence of the StAR mRNA in male and female adrenals, in tests and ovaries, but not in the liver or kidneys of either sex. Whole hamster adrenals revealed the presence of four mRNAs of 0.65, 1.7, 3.1 and 5.25 kb, respectively. In addition, ACTH regulates the expression of StAR mRNA in hamster adrenals. Indeed, when groups of hamsters were injected with ACTH and sacrificed at different times after treatment, only the 0.65 kb form of the StAR mRNA did not increase, whereas the other forms increased at varying levels. These results might suggest that the expression of the StAR protein in hamster adrenals depends upon different genes, different promoters, or different polyadenylation signal sites. In conclusion, these results indicate that in vivo, StAR is regulated by ACTH, suggesting the participation of this protein in controlling the transformation of cholesterol to pregnenolone, a key regulatory step in corticosteroidogenesis.

Adrenal Glands↗
PubMed1996

Characterization of the hamster CYP11B2 gene regulatory regions.

We have isolated a hamster CYP11B2 gene encoding the cytochrome P450 aldosterone synthase. In comparison with the CYP11B2 gene of other species, cis-elements named Ad1, Ad2, Ad3, and Ad4, were identified in the 5'-untranslated region of the hamster gene. Mouse adrenal tumor cells were transiently transfected with chimaeric reporter constructs, fused to the bacterial chloramphenicol acyltransferase (CAT) reporter gene, to study the regulation of expression of the hamster CYP11B2 gene. The highest basal expression was obtained with the -130 bp construct. Decreasing the length of the regulatory region of the CYP11B2 gene beyond that of -130 bp, to exclude Ad2 and Ad1 elements, resulted in successive decreases in CAT activity. Increasing the length of the regulatory region beyond that of -130 bp also resulted in a reduction of CAT activity, indicating the presence of inhibitory cis-elements in this area of the gene. Forskolin stimulated the CAT activity of all constructs, the highest of which occurred with the -130 bp construct, indicating that the gene is controlled by the PKA signalling pathway. TPA, however, had no stimulatory effects on any of these constructs. Staurosporine, an inhibitor of the PKC pathway, stimulated cells transfected with the different constructs in a similar manner as forskolin, indicating that PKC might act, at least in Y-1 cells, as a negative regulator on the hamster CYP11B2 promoter.

Adrenal Cortex Neoplasms↗
PubMed1996

Some effects of a low sodium intake on the expression of P450 aldosterone synthase in the hamster adrenal cortex: immunoblotting, immunofluorescent and immuno-gold electron microscopic studies.

In the current work we studied the effects of a low sodium intake on P450 aldosterone synthase (P450aldo) in the adrenal cortex of male hamsters by Western blotting analysis. We also investigated the zonal distribution of P450aldo with a specific antibody using immunofluorescence and immuno-gold electron microscopy. Western blotting analysis revealed a progressive induction of P450aldo in the adrenals of hamsters kept on a low sodium diet, with two-, four- and eightfold increases after 2, 4 and 21 days on the diet. Immunofluorescence microscopy showed that P450aldo was confined to the zona glomerulosa (ZG) cells. Electron microscopy showed P450aldo to be located in the mitochondria of ZG cells. When hamsters were maintained on a low sodium intake for 2, 11 and 21 days, P450aldo was still found only in the ZG; the ZG appeared either unchanged or sometimes slightly enlarged. Moreover, at days 11 and 21, the intensity of the immunofluorescent signal was much stronger in the ZG of hamsters on the low sodium intake than in controls. Hence, immunocytochemistry using the colloidal-gold technique showed P450aldo to be more abundant in the mitochondria of the experimental animals than in controls. To conclude, P450aldo is present only in the ZG of hamster adrenals and sodium restriction appears to induce its expression by stimulating production within individual ZG cells rather than by stimulating a proliferation of the ZG cells.

Adrenal Cortex↗
PubMed1995

Expression and characterization of isoforms of 3 beta-hydroxysteroid dehydrogenase/delta 5-->4-isomerase in the hamster.

The enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5-->4-isomerase (3 beta-HSD) is essential for the production of all classes of steroid hormones. Multiple isozymes of this enzyme have been demonstrated in the kidney and liver of both the rat and the mouse, although the function of the enzyme in these tissues is unknown. We have characterized three isozymes of 3 beta-HSD expressed in various tissues of the hamster. Both western and northern blot analyses demonstrated very high levels of 3 beta-HSD in the adrenal, kidney and male liver. Conversely, there were extremely low levels of enzyme expression in the female liver. cDNA libraries prepared from RNA isolated from hamster adrenal, kidney and liver were screened with a full-length cDNA encoding human type 1 3 beta-HSD. Separate cDNAs encoding three isoforms of 3 beta-HSD were isolated from these libraries. To examine the properties of the isoforms, the cDNAs were ligated into expression vectors for over-expression in 293 human fetal kidney cells. The type 1 isoform, isolated from an adrenal cDNA library, was identified as a high-affinity 3 beta-hydroxysteroid dehydrogenase. A separate isoform, designated type 2, was isolated from the kidney, and this was also a high-affinity dehydrogenase/isomerase. Two cDNAs were isolated from the liver, one identical in sequence to type 2 of the kidney, and a distinct cDNA encoding an isoform designated type 3. The type 3 3 beta-HSD possessed no steroid dehydrogenase activity but was found to function as a 3-ketosteroid reductase. Thus male hamster liver expresses a high-affinity 3 beta-HSD (type 2) and a 3-ketosteroid reductase (type 3), whereas the kidney of both sexes express the type 2 3 beta-HSD isoform. These differ from the type 1 3 beta-HSD expressed in the adrenal cortex.

Amino Acid Sequence↗
PubMed1994

The presence of two cytochrome P450 aldosterone synthase mRNAs in the hamster adrenal.

We isolated a cDNA from a hamster adrenal cDNA library which was similar in sequence to those of the mouse and rat P450c18 cDNAs. The hamster P450c18 cDNA, however, was shorter than the rat and mouse P450c18 cDNAs at its 5'-end and the peptide leader sequence was absent. From a hamster genomic library we isolated and sequenced the first seven exons and a 5'-flanking region of the first P450c18 gene exon. With this information we were able to generate a P450c18 cDNA containing the peptide leader sequence using the polymerase chain reaction. Northern analyses were performed on adrenals from hamsters maintained on a low sodium diet for 0, 4, 7 and 10 days using a 32P-labeled sequence specific to P450c18; two mRNA bands were found at 2 and 3.4 kb. The intensity of both bands was increased about 3- to 5-fold under sodium restriction compared to controls. A distinct mRNA band of 2.3 kb hybridized with an oligonucleotide specific to P450(11) beta and its intensity did not change following low sodium intake. Immunoblotting analyses were performed using an antibovine adrenal P450(11) beta antibody that does not discriminate between P450(11) beta and P450c18 proteins. Three bands were detected at 52, 48 and 45 kDa in homogenate preparations of entire glands. Furthermore, the 45 kDa protein band was present in homogenates of the zona glomerulosa and absent in homogenates of the zone fasciculata-reticularis. In conclusion, these results show that the hamster adrenals express P450c18 as do mouse, rat and human adrenal glands. Furthermore, two P450c18 mRNAs, which are inducible by a low sodium intake, are present in the hamster adrenal vs one for the rat. The physiological role of these two hamster adrenal mRNA species remains to be elucidated.

Adrenal Glands↗