Immunolocalization of a mesenchymal antigen specific to the gastrointestinal tract

Calvert, R.; Millane, Ghania; Beaulieu, Jean‐François

doi:10.1002/ar.1092400308

Cited by 8 publications

(5 citation statements)

References 44 publications

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“…After two washes, tissue sections were incubated for 2 h at room temperature with the respective anti-PKC␣, -⑀, or -antibodies (diluted in PBS containing 1% bovine serum albumin), and then washed twice. Tissues sections were then incubated for 30 min with a fluorescein-conjugated goat anti-rabbit IgG (Roche Molecular Biochemicals, Laval, Quebec, Canada) diluted 1/50, washed in PBS for 5 min, and then mounted in glycerol-PBS (9:1) containing 0.1% phenylenediamine (21). Control sections were incubated with the second antibody only.…”

Section: Methodsmentioning

confidence: 99%

Control of CYP11B2 Gene Expression through Differential Regulation of Its Promoter by Atypical and Conventional Protein Kinase C Isoforms

Lehoux

Dupuis

Lefèbvre

2001

Journal of Biological Chemistry

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-, or -did not inhibit these effects. Gö 6976 enhanced promoter activity, providing further evidence that PKC␣ was involved. Various CYP11B2 promoter constructs were used to identify the area associated with TPA and PKC inhibition. TPA and CA-PKC␣, -⑀, or -abolished the effects of AII, KCl, and Bt 2 cAMP on the activity of ؊102 and longer constructs. In summary, our findings suggest that the hamster CYP11B2 gene is under differential control by conventional (␣) and atypical () PKC.Aldosterone synthase is the product of the CYP11B2 gene and is responsible for the final step of aldosterone synthesis in most mammalian adrenal glands (1). This key regulatory enzyme is under the control of many factors including angiotensin II (AII), 1 adrenocorticotropin (ACTH), and changes in blood Na ϩ and K ϩ concentrations (1). The hamster adrenal CYP11B2 gene has been cloned and its promoter activity studied (2, 3). In transfected human adrenocortical carcinoma NCI-H295 cells, the first 161 base pairs of the 5Ј-flanking region of the hamster CYP11B2 were shown to be sufficient for maximal promoter activity and for the stimulatory effect of AII, KCl, and Bt 2 cAMP (4). In comparison, the first 129 base pairs of the 5Ј-flanking region of the human CYP11B2 gene were also shown to be responsive to the stimulatory action of AII, KCl and dibutyryl cyclic adenosine 3Ј:5Ј-monophosphate (Bt 2 cAMP) (5).The involvement of protein kinase C (PKC) in the expression of adrenal steroidogenic enzymes has been studied using activators and inhibitors of PKC. For example, 12-O-tetradecanoylphorbol-13-acetate (TPA) has been reported to increase the mRNA levels of 3-␤-hydroxysteroid dehydrogenase (6) and cytochrome P450 21-hydroxylase in the NCI-H295 cell line (7,8). In addition, phorbol esters have been shown to decrease the mRNA levels of cytochrome P450 17␣-hydroxylase (7) and cytochrome P450 side chain cleavage (7) and to abolish the enhancing effect of BAYK 8644 on cytochrome P450 11␤-hydroxylase (P450C11) and cytochrome P450 aldosterone synthase (P450aldo) (9). Steroidogenesis is also affected by inhibition of PKC. For instance, staurosporine has been reported to stimulate steroidogenesis in Y-1 cells (3, 10), to elevate the levels of mRNA of cytochrome P450 side chain cleavage and P450C11 (10), and to stimulate the activity of the hamster CYP11B2 promoter in transfected Y-1 cells (3). We have reported that GF109203X, a specific PKC inhibitor, has stimulated the output of aldosterone and the activity of the hamster CYP11B2 promoter in NCI-H295-transfected cells (4). In addition, staurosporine has been reported to reverse the inhibitory effect of TPA on aldosterone production in rat adrenal glomerulosa cells and to enhance the stimulatory effects of AII and K ϩ , but not of ACTH, in these cells (11).PKC comprises several isoforms of serine/threonine kinases that can be divided in three families (12)(13)(14). Members of the conventional family (PKC␣, -␤1, -␤2, and -␥) are calcium-dependent and are activated by phosphatidylserine and diacylglyce...

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Section: Methodsmentioning

confidence: 99%

Control of CYP11B2 Gene Expression through Differential Regulation of Its Promoter by Atypical and Conventional Protein Kinase C Isoforms

Lehoux

Dupuis

Lefèbvre

2001

Journal of Biological Chemistry

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“…The slides were then incubated for 30 min with a fluorescein-conjugated goat antirabbit IgG (Roche Molecular Biochemicals, Mannheim, Germany) diluted 1:50, washed in PBS for 5 min, and then mounted in glycerol-PBS (9:1) containing 0.1% phenylenediamine (29). For the localization of StAR, adrenal glands were excised from three different animals in each experimental group.…”

Section: Immunolocalizationmentioning

confidence: 99%

Thein VivoEffects of Adrenocorticotropin and Sodium Restriction on the Formation of Different Species of Steroidogenic Acute Regulatory Protein in Rat Adrenal¹

et al. 1999

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We have studied the in vivo expression of steroidogenic acute regulatory protein (StAR) in adrenals of control, ACTH-treated, and Na+-restricted rats. Indirect immunofluorescence by microscopy revealed the presence of StAR in the zonae glomerulosa (ZG) and fasciculata-reticularis (ZFR). An increased signal was observed in the ZG and zona fasciculata, 5 h after ACTH injection; a few cells of the medulla were also positive. Immunogold electron microscopy showed that StAR was mainly located over mitochondria (MT). By immunoblotting, a major 29-kDa and other minor StAR bands migrating between 30 and 39 kDa were increased 5 h after ACTH treatment but remained unchanged after 1 h. By two-dimensional-PAGE, four StAR species were revealed in homogenates of control ZG, and their intensity was increased 5 h after ACTH treatment but not after 1 h. Also, additional acidic species were seen 5 h after treatment. Other bands with basic isoelectric point were revealed between 29 and 37 kDa. Analyses on whole gland MT and supernatant (SN) revealed four bands in the control SN and five in ACTH SN; the intensity of one band was increased, and that of another one was decreased, in SN of treated rats. ACTH treatment resulted in the localization of many low-isoelectric point StARs in MT. After two-dimensional-PAGE, differences were found in the mobility of some StAR species in the ZG between controls and Na+-restricted rats. In MT, four bands were revealed in the ZG preparations of Na+-restricted and two bands in controls. Four bands were revealed in the ZG SNs of control and Na+-restricted rats; an additional band was observed only in the SN of treated animals, whereas the intensity of another band decreased. Na+ restriction did not affect StAR in the ZFR. In conclusion, StAR was present in the rat adrenal cortex ZG and ZFR and was mainly located in MT. StAR expression was inducible in the ZG and the ZF by ACTH, resulting in the formation of many StAR acidic species; interestingly, such changes were detectable 5 h, but not 1 h, after ACTH administration, suggesting that steroidogenesis stimulation by StAR might occur mainly outside MT. Although less spectacular than for ACTH, Na+ restriction also affected StAR expression in the ZG but not in the ZFR, by increasing two mitochondrial and one SN species, implying that StAR is involved in the mechanism of action of Na+ restriction in promoting aldosterone formation. These results suggest that differential processing and/or changes in phosphorylation may occur in vivo upon ACTH treatment and Na+ restriction. We hypothesize that modification of a relatively small quantity of StAR, mainly located outside MT, is necessary to increase adrenal steroidogenesis challenged either by ACTH or Na+ restriction.

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“…After two washes, tissue sections were incubated for 2 hr at room temperature (RT) with the rabbit anti-rat adrenal P450C17 antibody (diluted 1:100 in PBS containing 1% BSA) and then washed twice. They were next incubated for 30 min with a fluorescein-conjugated goat anti-rabbit IgG (Boehringer; Mannheim, Germany) diluted 1:50, washed in PBS for 5 min, and then mounted in glycerol-PBS (9:1) containing 0.1% phenylenediamine (Calvert et al 1994). The tissue sections were studied with a Reichert Polyvar 2 microscope equipped for epifluorescence.…”

Section: Immunofluorescencementioning

confidence: 99%

Immunolocalization and Biochemical Determination of Cytochrome P450C17 in Adrenals of Hamsters Treated with ACTH

Brière

Martel

Cloutier

et al. 1997

J Histochem Cytochem.

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SUMMARYWe 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.

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Immunolocalization of a mesenchymal antigen specific to the gastrointestinal tract

Cited by 8 publications

References 44 publications

Control of CYP11B2 Gene Expression through Differential Regulation of Its Promoter by Atypical and Conventional Protein Kinase C Isoforms

Control of CYP11B2 Gene Expression through Differential Regulation of Its Promoter by Atypical and Conventional Protein Kinase C Isoforms

Thein VivoEffects of Adrenocorticotropin and Sodium Restriction on the Formation of Different Species of Steroidogenic Acute Regulatory Protein in Rat Adrenal¹

Immunolocalization and Biochemical Determination of Cytochrome P450C17 in Adrenals of Hamsters Treated with ACTH

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Immunolocalization of a mesenchymal antigen specific to the gastrointestinal tract

Cited by 8 publications

References 44 publications

Control of CYP11B2 Gene Expression through Differential Regulation of Its Promoter by Atypical and Conventional Protein Kinase C Isoforms

Control of CYP11B2 Gene Expression through Differential Regulation of Its Promoter by Atypical and Conventional Protein Kinase C Isoforms

Thein VivoEffects of Adrenocorticotropin and Sodium Restriction on the Formation of Different Species of Steroidogenic Acute Regulatory Protein in Rat Adrenal1

Immunolocalization and Biochemical Determination of Cytochrome P450C17 in Adrenals of Hamsters Treated with ACTH

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Thein VivoEffects of Adrenocorticotropin and Sodium Restriction on the Formation of Different Species of Steroidogenic Acute Regulatory Protein in Rat Adrenal¹