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E N Cozza

Publications and source records attributed to E N Cozza.

36 records · Page 2Linked to original sources
PubMed1993

Synthesis of 18-hydroxycortisol and 18-oxocortisol in bovine adrenal zona glomerulosa mitochondria.

The biosynthesis of 18-hydroxycortisol and 18-oxocortisol from cortisol was studied in calf adrenal zona glomerulosa mitochondria. Cortisol is converted to 18-hydroxycortisol and 18-oxocortisol in the same mitochondrial preparation in which corticosterone is metabolized to 18-hydroxycorticosterone and aldosterone. Cortisol and 18-hydroxycortisol interacted with mitochondria to cause a Type I differential spectrum, which was decreased by sodium dithionite. The metabolism of cortisol to 18-hydroxycortisol and 18-oxocortisol was inhibited by metyrapone in a competitive way. Cortisol was a competitive inhibitor of the transformation of corticosterone into 18-hydroxycorticosterone and aldosterone, and corticosterone was a competitive inhibitor of the transformation of cortisol into 18-hydroxycortisol and 18-oxocortisol, with a Ki very similar to the Km for the transformation of that steroid to aldosterone. These results indicate that cortisol is metabolized to 18-hydroxycortisol and 18-oxocortisol by a mitochondrial cytochrome P-450, which is the same as that which catalyzes the conversion of corticosterone into aldosterone.

18-Hydroxycorticosterone↗
PubMed1992

Endothelin-1 potentiation of angiotensin II stimulation of aldosterone production.

Endothelin-1 (ET-1) binds to specific receptors in cultured bovine adrenal glomerulosa cells and stimulates aldosterone secretion with a 50% effective concentration (EC50) of 300 +/- 80 pM (mean +/- SE). The relative stimulatory potency for ET-1 is significantly less than that of angiotensin II (ANG II). The incubation of calf zona glomerulosa cells in primary culture with ET-1 and ANG II resulted in a significant potentiation of ANG II effect on aldosterone secretion. The EC50 of ET-1 potentiation of ANG II-induced stimulation of aldosterone secretion was 40 +/- 5 pM (mean +/- SE, n = 4), which is lower than the EC50 for ET-1 stimulation of aldosterone secretion. Adrenocorticotropic hormone (ACTH) stimulation of aldosterone secretion, but not that of potassium, was also potentiated by ET-1, but to a lesser degree. ET-1 and ET-1-mediated potentiation of ANG II-stimulated aldosterone biosynthesis increased both the early and late pathways of aldosterone biosynthesis, but the potentiation was greater for the early pathway. Preincubation with ET-1 for at least 15 min, followed by extensive washing to remove bound ET-1, also resulted in persistent potentiation of ANG II-mediated aldosterone secretion. ET-2, sarafotoxin, and vasoactive intestinal contractor potentiation of ANG II action were very similar to that of ET-1. ET-3 and Big-ET-1 potentiated ANG II stimulation only at the highest doses tested and the proendothelin-(110-130) fragment was inactive. ET-1 potentiation of ANG II action is likely to be mediated through an ETB receptor subtype.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone↗
PubMed1990

ET-1 receptors in C-6 cells: homologous down-regulation and modulation by protein kinase C.

Endothelin (ET-1) receptors were studied in the C-6 glia cell line. ET-1 binds to C-6 cells in a temperature- and time-dependent manner, with an apparent Kd of 1.16 +/- 0.07 10(-10) M and a Bmax of 96,500 +/- 6000 sites/cell (mean +/- SEM, n = 27). Stimulation of protein kinase C (PKC) with the diacylglycerol (DAG) analog phorbol 12-myristate 13-acetate (PMA) resulted in a decrease in the number of receptors in a dose-dependent manner. Inhibition of PKC with H-7 eliminated the effect of PMA on the reduction of binding sites. Treatment with exogenous 1-oleoyl-2-acetyl-sn-glycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DOG), release of endogenous DAG with phospholipase C, and inhibition of the metabolism of DAG with the diacylglycerol kinase inhibitor R 59022 also resulted in a decrease in the number of receptors. The effect of these agents was inhibited by H-7. ET-1-mediated down-regulation of receptors was also demonstrated, but the down-regulation was not affected by H-7 or by depletion of cellular PKC with chronic, high dose of PMA. Internalization constants of ET-1-receptor complex was also measured according to the model of Wiley and Cunningham (Cell 25 (1981) 433). PMA- and ET-1-mediated down-regulation of receptors was associated with an increase in the endocytosis constant for the hormone-receptor complex and a decrease in the rate of insertion of receptor into the plasma membrane. PMA, but not ET-1, increased the rate of endocytosis of unoccupied receptors. Radioiodinated ET-1 was crosslinked to the receptor after binding, extracted and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A band at 66 kDa was obtained. These studies show that ET-1 and PKC activation produce down-regulation of ET-1 membrane receptors and that ET-1-mediated down-regulation probably does not involve the activation of PKC.

Animals↗
PubMed1990

The binding of cortisol to adrenal mitochondria.

Binding of tritiated cortisol to adrenal zona glomerulosa mitochondria was studied and compared with that of corticosterone. Cortisol was shown to bind specifically to the inner membrane of zona glomerulosa mitochondria. Corticosterone and cortisol had similar apparent association constants (Ka) and concentrations of binding sites. The methodology was validated by obtaining similar Ka from both binding plots and kinetic data. Cortisol binding was inhibited by pretreatment with sodium dithionite, and displaced by deoxycorticosterone, corticosterone, 18-hydroxy-corticosterone, 11 beta-hydroxy-18-ethynyl-progesterone and metyrapone, but not by cholesterol. These results suggest that cortisol and corticosterone bind to the same cytochrome P-450.

Animals↗
PubMed1990

ACTH increases de novo synthesis of diacylglycerol and translocates protein kinase C in primary cultures of calf adrenal glomerulosa cells.

Effects of ACTH on the production of diacylglycerol (DAG) and translocation of protein kinase C were studied in primary cultures of calf adrenal glomerulosa cells. To study DAG production two different labeling protocols were used: (a) cells were prelabeled for 3 days with [2-3H]glycerol before ACTH addition; (b) ACTH and [2-3H]glycerol were added simultaneously to cells. In both cases, ACTH provoked rapid increases in the labeling of DAG which were maximal in 2 min, dose-dependent, and paralleled by increases in DAG mass. ACTH also increased the labeling of total glycerolipids including phosphatidic acid (PA), phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine and triacylglycerol. In both labeling protocols, the rates of increase in the labeling of DAG and PA were greater than those of other glycerolipids. Our results indicate that ACTH rapidly increases DAG, at least partly by stimulating the de novo synthesis of PA. In addition, we found that ACTH, like phorbol esters, stimulated the apparent translocation of immunoreactive protein kinase C from the cytosol to the membrane fraction.

Adrenocorticotropic Hormone↗
PubMed1990

Endothelin receptor subtypes and stimulation of aldosterone secretion.

Endothelins (ETs) are 21-amino acid peptides with two disulfide bonds that have powerful vasoactive properties. We have previously shown the presence of a specific, high-affinity, saturable receptor for porcine or human endothelin (ET-1) in cultured calf zona glomerulosa cells. ET-1 was a stimulator of aldosterone secretion although not as powerful as angiotensin II. Incubations of cultured calf zona glomerulosa cells with Sarafotoxin S6b (S6b), a snake venom that has a structure highly homologous to ET-1, stimulated aldosterone secretion with a potency similar to that of ET-1. Binding of [125I]ET-1 to the adrenal receptor gave a Kd of 0.17 +/- 0.05 nM and a Bmax of 36 +/- 8.5 fmol/well (n = 4). Displacement of [125I]ET-1 by unlabeled ETs and S6b showed that the concentrations needed to displace 50% of the tracer were 0.3 nM for ET-1, 0.3 nM for ET-2, 10 nM for S6b, and 100 nM for ET-3. Binding of [125I]S6b to cultured adrenal cells revealed a receptor with a Kd of 0.05 +/- 0.01 nM and a Bmax of 8 +/- 2 fmol/well (n = 4). Displacement of [125I]S6b by unlabeled ETs and S6b showed that the concentrations needed to displace 50% of the tracer were 0.03 nM for S6b, 0.06 nM for ET-1, 0.04 nM for ET-2, and 0.05 nM for ET-3. Unlabeled ET-1 and ET-2 preferentially down-regulated the binding of [125I]ET-1, and S6b preferentially down-regulated the binding of [125I]S6b.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldosterone↗
PubMed1990

Treatment of primary cultures of calf adrenal glomerulosa cells with adrenocorticotropin (ACTH) and phorbol esters: a comparative study of the effects on aldosterone production and ACTH signaling system.

We studied the mechanism that underlies the desensitization of calf adrenal glomerulosa cells induced by 4 h of ACTH treatment. In control cells, acute ACTH treatment provoked sizeable increases in aldosterone, cAMP, and diacylglycerol, and translocated protein kinase-C from cytosol to membrane. In desensitized cells, acute ACTH effects on aldosterone and cAMP decreased by 25-60%, and diacylglycerol levels and protein kinase-C translocation were persistently stimulated and not substantially affected by further acute ACTH treatment. After 4 h of treatment with 1 microM phorbol 12-myristate 13-acetate (PMA) there were no acute effects of ACTH on the production of aldosterone, cAMP, or diacylglycerol or on protein kinase-C, which was already strongly translocated. These results suggest that ACTH-mediated desensitization of calf adrenal glomerulosa cells may be at least partially mimicked by long term treatment with phorbol esters and could be due to ACTH-induced increases in diacylglycerol-protein kinase-C signaling.

Adrenal Glands↗
PubMed1990

Effects of endothelin-1 on its receptor concentration and thymidine incorporation in calf adrenal zona glomerulosa cells: a comparative study with phorbol esters.

Endothelin (ET-1) is a 21-amino acid peptide with potent vasopressor and vasoconstrictive properties. Specific, high affinity receptors for ET-1 have been found in the adrenal gland. The stimulation by ET-1 of aldosterone secretion in cultured calf zona glomerulosa cells was shown to depend on the serum used for culturing and was not related to the growth-promoting effects of serum or the response to another secretagogue, such as angiotensin-II. In this study, binding of [125I]ET-1 to crude membrane preparations from calf adrenal cortex slices showed that ET-1 binding was greater in the outer slices, corresponding to the zona glomerulosa, than in inner slices, corresponding to the zona fasciculata. ET-1 stimulated aldosterone, but not cortisol, biosynthesis. Adrenal zona glomerulosa preincubated with ET-1 resulted in homologous down-regulation. Since ET-1 action involves activation of protein kinase-C (PKC), we studied the effect of a phorbol ester (PMA) on the down-regulation of ET-1 receptors. PMA decreased the number of cell surface receptors, and its effect was prevented by pretreatment with the PKC inhibitors H-7 and sphyngosine. Agonist-mediated down-regulation could not be blocked by pretreatment with PKC inhibitors, suggesting that PKC is involved in phorbol ester-mediated, but not agonist-mediated down-regulation of ET-1 receptors. Both effectors increased the endocytosis rate constant as well as the steady state cytosolic membrane-bound ratio for ET-1 receptors, suggesting that the decrease in the number of cell surface receptors is at least partially due to an increased internalization of the hormone-receptor complex. ET-1 and PMA decreased the incorporation of [3H]thymidine into calf zona glomerulosa cell cultures. We conclude that ET-1 and PMA have similar effects on glomerulosa cells, producing down-regulation of ET-1 receptors and an antimitogenic effect, but these actions are through different mechanisms.

Aldosterone↗
PubMed1989

Effects of ACTH on the last step of aldosterone biosynthesis.

The production of tritiated aldosterone and tritiated SM (a saponifiable 18-hydroxycorticosterone derivative) by rat adrenals were studied at various incubation times in absence or presence of two concentrations of ACTH. Tritiated 18-hydroxycorticosterone or 18-deoxyaldosterone served as precursors. The lower ACTH concentration (150 pM) increased the production of tritiated aldosterone. Whereas, the higher ACTH concentration (1.5 microM) stimulated tritiated aldosterone production at shorter incubation time (30 min), while after 60 min it inhibited. This time dependency would reflect variations in the levels of endogenous steroids. On the other hand, the effects of ACTH on tritiated SM production were opposite to those on tritiated aldosterone. In effect, while 150 pM ACTH inhibited SM production, 1.5 microM ACTH stimulated it. These results suggest that ACTH promotes opposite effects on the productions of aldosterone and SM and therefore both productions would be coordinated under the regulation of ACTH.

Adrenal Glands↗
PubMed1989

Endothelin binding to cultured calf adrenal zona glomerulosa cells and stimulation of aldosterone secretion.

Endothelins are a group of potent vasoconstrictors whose structure was deduced from genomic DNA. ET-1 was first isolated from culture supernatants from porcine endothelial cells and ET-3 was identified from a rat DNA library. We report on the binding of 125I-ET-1 to zona glomerulosa cells in culture and on its ability to stimulate aldosterone secretion. Cultured calf adrenal zona glomerulosa cells have saturable, high affinity [Kd = 1.00 +/- 0.17 X 10(-10) M (SEM)] receptors which bind ET-1 in a temperature and time dependent manner. Binding was specific and angiotensin II, vasopressin, ANP, BNP, apamin, calcium channel agonists or antagonists did not interact with the receptor. ET-3 displaced 125I-ET-1 from the receptor with a relative potency of 0.39 +/- 0.1% (SEM) that of ET-1. ET-1 incubated with cultured glomerulosa cells stimulated aldosterone secretion in a dose dependent manner but it was less potent than angiotensin II. ET-3 had less than 1% the relative potency of ET-1 stimulating aldosterone secretion. This data suggest that ET-1 is an independent stimulator of aldosterone secretion and we are speculating that it might be important in those situations, like in malignant hypertension, where endothelial damage might result in increased ET-1 production.

Aldosterone↗
PubMed1988

ACTH stimulates turnover of the phosphatidylinositol-glycan.

Primary cultures of calf adrenal glomerulosa cells were prelabeled for 3 days with [3H]inositol or [3H]glucosamine and stimulated with 10 nM ACTH. Labeled phosphatidylinositol (PI), polyphosphoinositides (PIP and PIP2) and a novel phosphatidylinositol-glycan (PI-glycan) were measured after separation by TLC. [3H]-Inositol labeling of PI, PIP and PIP2 increased rapidly, whereas labeling of the PI-glycan showed an initial decrease at 1 minute followed by a subsequent increase. Similar results were obtained when cells were prelabeled with [3H]glucosamine, viz. the PI-glycan label decreased at 1 min and subsequently increased. These results suggest that ACTH provokes (a) coordinated increases in the synthesis of PI, PIP, PIP2 and the PI-glycan, and (b) the increase in PI-glycan synthesis is preceded by initial decrease, presumably reflecting hydrolysis of this lipid.

Adrenocorticotropic Hormone↗
PubMed1987

Versatile steroid molecules at the end of the aldosterone pathway.

18-hydroxycorticosterone converts spontaneously and reversibly to a variety of less polar forms and derivatives, some of which are precursors to aldosterone. In particular, 21-hydroxy-11 beta, 18-oxido-4-pregnene-3,20-dione (18-DAL) is hydroxylated to aldosterone with high yields in the presence of malate and NADP+, at pH 4.8. 18-DAL also behaves as a metabolic intermediate between 18-OH-B and aldosterone according to time-course and trapping experiments. Consequently, the final steps of the aldosterone pathway at pH 4.8 could be identified as 18-OH-B, 18-DAL and aldosterone, in this sequence. The submitochondrial distribution of aldosterone biosynthesis is compatible with this postulate. The work also shows that some forms of 18-OH-B are promoters of hydrogen transport in renal tubuli and that this regulation may be independent of sodium reabsorption. These results suggest a regulatory model, new in steroid biology, according to which steroid molecules bearing an oxidized angular C18-methyl may undergo structural changes between precursor ("P") and hormonal ("H") forms in response to homeostatic requirements.

18-Hydroxycorticosterone↗
PubMed1987

Convertibility of a saponifiable lipoidal derivative of 18-hydroxycorticosterone.

Metabolic properties and subcellular localization of the biosynthesis of SM, a saponifiable 18-OH-B (18-Hydroxycorticosterone) derivative, were investigated. Homogenates biosynthesized SM at a nearly constant rate of 463 pmol/50 mg tissue during 30 min. This biosynthesis was more efficient at pH 7.4 than at pH 4.8. Not only 18-OH-B but also its less polar anhydride 18-DAL (18-Deoxyaldosterone) were good precursors. SM was reverted to these precursors both enzymatically and spontaneously, 4.8 being a more suitable pH for this reversion than 7.4. Trapping experiments demonstrated a sequence comprising, in this order, the following echelons: SM, 18-OH-B, 18-DAL, Aldosterone. The first two steps are reversible and the last two ones depend on proton concentrations. It is postulated that SM could be on a dead-end to which 18-OH-B could be deviated if Aldosterone biosynthesis became temporarily unnecessary. Also, that 18-OH-B may convert to either 18-DAL or SM for selective membrane transports, according to homeostatic requirements.

18-Hydroxycorticosterone↗
PubMed1985

Eighteen-deoxyaldosterone and other less polar forms of 18-hydroxycorticosterone as aldosterone precursors in rat adrenals.

Samples containing as precursors either 18-hydroxycorticosterone (18-OH-B) in its M form, or this converted to less polar forms at pH 2 (ACM), or M or ACM enclosed in liposomes from adrenal lipids were incubated at pH 7.4, 4.8 or 3.3 in the presence or absence of quartered rat adrenals for 1 and 2 h. Optimal (10%) yields of aldosterone were obtained when (a) ACM was incubated at pH 4.8 and (b) M enclosed in liposomes was suspended in buffer and shaken without enzyme at pH 3.3. When conditions (a) were supplemented with malate and NADP, 16% of ACM was converted to aldosterone. ACM contained 80% of a fraction which, according to 13C NMR spectroscopy, was identical to 18-deoxyaldosterone (18-DAL). Experiments in which radioactivity from corticosterone (B) or M was trapped by radioinert M or 18-DAL disclosed a pathway comprising sequentially B, 18-OH-B, 18-DAL and aldosterone, and the combined evidence of this work, an enzymatic hydroxylation of 18-DAL to aldosterone.

18-Hydroxycorticosterone↗
PubMed1983

Progesterone and its reductive metabolism in steroidogenic tissues of the developing hen embryo.

Progesterone contents of adrenals and ovaries and the--mainly reductive--metabolism of [3H]progesterone by these organs and liver were investigated in hen embryos between Days 13 and 21 (hatching). Progesterone contents are similar in adrenals and ovaries on Day 13 (approx 3.5 ng/mg) but descend in characteristic manners toward Day 17 and rise steeply, only in the ovaries, then descend in these organs toward hatching. [3H]Progesterone is converted by the adrenals toward 12 main metabolites, the main glucocorticoid being corticosterone (B), and the main reduced metabolite, 5 beta-pregnane-3,20 dione (5 beta-P). On day 13, 5 beta-P is five times as important as B, but both steroids evolve in a symmetric fashion, so that at hatching this proportion is reversed. In all tissues at all stages, except the liver on Day 13, the yields of 5 alpha-pregnane-3,20 dione (5 alpha-P) are one order of magnitude below those of 5 beta-P. Both diones exhibit maxima on Day 17, probably extending until Day 19. Concomitantly, [3H]progesterone disappearance is maximal on Day 17. Both ovaries differ in the shape of their 5 alpha-P/5 beta-P curves in that the left ovary exhibits for this curve a function ascending continuously toward hatching.

Adrenal Glands↗
PubMed1983

Corticoidogenesis in Bufo arenarum H. I. In vitro biosynthesis of [3H]pregnenolone and [3H]corticosterone metabolites and of endogenous 3-oxo-4-ene intermediates at 28 degrees C and 37 degrees C.

Sliced interrenal tissue from Bufo arenarum H. was incubated without exogenous substrate (to determine endogenous corticosterone (B) and progesterone), as well as in the presence of [3H]pregnenolone ([3H]Pre) and [3H]corticosterone, at 28 and at 37 degrees C. When yields of radiometabolites were analyzed as such, [3H]Pre at 28 degrees C was found to be a good precursor for 18-hydroxycorticosterone (18-HO-B), aldosterone (aldo), and an unknown N very similar but not equal to 19-hydroxycorticosterone. However, [3H]Pre at 28 degrees C was not a good precursor for corticosterone. When the same tritiated substrate was incubated at 37 degrees C, the yields of 18-HO-B and N diminished significantly, aldo remained as it was at 28 degrees C, and B increased. [3H]B was a poor precursor to aldo, 18-HO-B, and N, irrespective of incubation temperatures, but a good precursor to material with the mobility of 11-dehydro-B and an aldo-like compound X. Measurement of endogenous B and progesterone pools followed by calculations of specific activities lead to the conclusion that these differences should not be ascribed to artifactual competitions between precursor pools for enzyme systems. This species is thus characteristic in its capacity to bypass, at least at 28 degrees C and under the in vitro conditions employed, corticosterone during aldo biosynthesis. Furthermore, the vast ambient temperature ranges to which this and many other amphibian species are exposed and the temperature dependence observed in corticoidogenesis of B. arenarum suggest that corticoidogenic studies in these species should be carried out at more than one temperature.

18-Hydroxycorticosterone↗
PubMedDate not supplied

Cortisol: a tool to study aldosterone biosynthesis in rats.

Corticosterone (B) and 18-hydroxy-11-deoxycorticosterone (18OHDOC) but not 11-deoxycorticosterone (DOC) displaced cortisol (F) specifically bound to rat adrenal mitochondria. F. competitively inhibited aldosterone formation from B, 18OHB and 18OHDOC but did not inhibit conversions of DOC to B or 18OHDOC. High concentrations of DOC increased its conversion to 18OHDOC rather than B.

18-Hydroxydesoxycorticosterone↗