scholarly journals Guinea pig adrenocortical cells: in vitro characterization of separated zonal cell types.

1982 ◽  
Vol 94 (2) ◽  
pp. 241-252 ◽  
Author(s):  
P Miao ◽  
V H Black
Keyword(s):  
Guinea Pig ◽  
Morphological Characteristics ◽  
Zona Glomerulosa ◽  
Equilibrium Density ◽  
Zona Fasciculata ◽  
Adrenocortical Cells ◽  
Steroid Production ◽  
Glomerulosa Cells ◽  
Zona Glomerulosa Cells

This paper reports a quick, relatively simple and reproducible technique for obtaining populations of zona fasciculata and zona glomerulosa cells up to 80-90% pure, which can be maintained in vitro for study of adrenocortical cell function. Isolated guinea pig adrenocortical cells were separated on a 1-28% bovine serum albumin/Ca++, Mg++-free buffer gradient (wt/vol at 4% increments) using equilibrium density centrifugation (570 g, 30 min). Over 60% of the 8 x 10(5) viable cells/adrenal obtained in the total isolate were recovered after separation. 80% of the zona glomerulosa cells were found in the lower three bands of the gradient. 78% of the zona fasciculata cells were found in the top three bands. Of the cells in the first two bands, 78-91% were zona fasciculata cells, whereas of the cells in the bottom two bands 92-95% were zona glomerulosa cells. The cells retained the morphological characteristics of cells in situ and could be maintained in vitro for periods up to 11 d. They produced a wide variety of steroids, cortisol, corticosterone, aldosterone, 11-beta-hydroxyandrostenedione, deoxycortisol, deoxycorticosterone, cortisone, 18-hydroxycorticosterone, and a product tentatively identified as dehydroepiandrosterone, and they responded to ACTH in a dose-responsive manner with enhanced levels of steroid output. Zona glomerulosa-enriched populations differed from zona fasciculata-enriched populations in their abundant production of aldosterone and in the pattern of steroid production. None of the cultures responded to angiotensin II (100 pg/ml) with increased steroid production.

Inhibition of corticosteroid production by sodium pentobarbitone in rat adrenocortical preparations

1993 ◽  
Vol 136 (1) ◽  
pp. 75-83 ◽  
Author(s):  
B. J. Whitehouse ◽  
S. J. Purdy ◽  
D. R. E. Abayasekara
Keyword(s):  
Intact Animal ◽  
Zona Glomerulosa ◽  
Zona Fasciculata ◽  
Steroid Production ◽  
Adrenal Cells ◽  
Glomerulosa Cells ◽  
Pituitary Adrenal Axis ◽  
Zona Glomerulosa Cells ◽  
Stimulation Of

ABSTRACT It is possible that some of the effects of sodium pentobarbitone on the hypothalamo-pituitary-adrenal axis in the intact animal may be attributable to direct actions on the adrenal cortex. The effects of the barbiturate on steroid production by rat adrenal preparations in vitro have therefore been examined. In zona glomerulosa cells, pentobarbitone inhibited basal steroid production in a dose-related fashion. For aldosterone and corticosterone, the doses required for 50% inhibition of production (IC50) were 1·2 mmol pentobarbitone/l and 3·7 mmol/l respectively. Steroidogenesis was inhibited at lower levels of pentobarbitone in the presence of 1 nmol ACTH/l (IC50 = 0·5 mmol pentobarbitone/l for aldosterone and 2·2 mmol/l for corticosterone). In zona fasciculata/reticularis cells, production of corticosterone was similarly reduced with an IC50 of 2·8 mmol pentobarbitone/l for basal production and 1·3 mmol/l for ACTH-stimulated production. The dose-related increases in corticosterone production produced by ACTH (0·1–1000 pmol/l) or dibutyryl cyclic AMP (0·1–1·0 mmol/l) were also eliminated in the presence of 2 mmol pentobarbitone/l. The effects of pentobarbitone (1–4 mmol/l) on the production of pregnenolone and deoxycorticosterone (DOC) were also studied. In zona fasciculata/reticularis cells, the responses of both pregnenolone and DOC were bell-shaped with increases at 1 mmol pentobarbitone/l, which fell back to control levels at 4 mmol pentobarbitone/l. Stimulation of DOC, accompanied by decreases in aldosterone and corticosterone production, was also seen in zona glomerulosa cells at 1 mmol pentobarbitone/l. The effect of 1 mmol pentobarbitone/l on the conversion of 22(R)-hydroxycholesterol (5-cholestene-3β,22(R)-diol), pregnenolone, progesterone and DOC to corticosterone and aldosterone by zona glomerulosa preparations was studied. There was a comparable reduction in the conversion of these precursors (2 μmol/l) to aldosterone with yields decreased to 20–30% of those found in the absence of pentobarbitone. The dose required for 50% reduction of the conversion of progesterone (2 μmol/l) to aldosterone was 0·55 mmol pentobarbitone/l and for corticosterone the dose was 1·75 mmol pentobarbitone/l. The results obtained show that pentobarbitone is an effective inhibitor of corticosteroid biosynthesis in rat adrenal cells, and suggest that its effects are brought about by inhibition of cytochrome P450-mediated hydroxylations. Journal of Endocrinology (1993) 136, 75–83

Potassium-induced aldosterone biosynthesis in cultured rat zona glomerulosa cells

1989 ◽  
Vol 256 (4) ◽  
pp. E475-E482
Author(s):  
J. Muller ◽  
M. Lauber ◽  
C. Schmid
Keyword(s):  
Molecular Mechanisms ◽  
Primary Cultures ◽  
Zona Glomerulosa ◽  
Zona Fasciculata ◽  
High Potassium ◽  
Primary Monolayer ◽  
Glomerulosa Cells ◽  
Potassium Restriction ◽  
Zona Glomerulosa Cells

Rat adrenal zona glomerulosa cells lost their ability to produce aldosterone from either endogenous precursors or added deoxycorticosterone within 2 days of primary monolayer culture in a medium with a potassium concentration of 6.3 mmol/l. The lost corticosterone methyl oxidase I and II activities were totally regenerated when the ambient potassium concentrations was raised to 31 mmol/l. The conversions of deoxycorticosterone to 18-hydroxycorticosterone and aldosterone were completely restored by culture in a high-potassium medium also in zona glomerulosa cells of rats in which aldosterone biosynthesis had been suppressed by potassium restriction and sodium loading. However, these conversions were not induced in zona fasciculata-reticularis cells. The induction of aldosterone biosynthesis was associated with the appearance of a mitochondrial 49,000 protein cross-reacting with an antibody raised against bovine adrenal cytochrome P-450(11) beta. Thus primary cultures of zona glomerulosa cells are promising models for studying in vitro the molecular mechanisms of long-term adaptation of aldosterone biosynthesis to sodium and potassium intake.

Relation of Intracellular K+ and Steroidogenesis in Isolated Adrenal Zona Glomerulosa and Fasciculata Cells

1975 ◽  
Vol 49 (1) ◽  
pp. 13-26
Author(s):  
F. A. Mendelsohn ◽  
C. Mackie
Keyword(s):  
Potassium Concentration ◽  
Zona Glomerulosa ◽  
Zona Fasciculata ◽  
High Potassium ◽  
External Potassium ◽  
Glomerulosa Cells ◽  
Unit Gravity Sedimentation ◽  
Zona Glomerulosa Cells ◽  
Stimulation Of

1. Intracellular K+ content, water spaces and corticosterone output were measured in isolated zona glomerulosa and zona fasciculata-reticularis cell suspensions of rat adrenal cortex, after incubations in vitro under conditions designed to alter steroidogenesis. 2. Intracellular K+ of unpurified zona glomerulosa cells was not altered after stimulation of corticosterone output with serotonin. Similarly, with zona glomerulosa cells purified by unit gravity sedimentation, no change in intracellular K+ was detected after stimulation of steroidogenesis with serotonin or angiotensin II. 3. In high-potassium medium (final concentration 84 mmol/l), parallel increases in intracellular K+ and corticosterone output were observed with both unpurified and purified zona glomerulosa cells. However, a similar increase in intracellular K+ also occurred in high-potassium medium with zona fasciculata cells, whose steroid output is unresponsive to external potassium concentration ([K+]). 4. Ouabain at 10−5 mol/l depressed the intracellular [K+] of glomerulosa cells but did not alter basal or stimulated corticosterone output. Similar results were obtained with fasciculata cells. 5. Ouabain at 5×10−4 mol/l further depressed intracellular [K+] of glomerulosa cells and inhibited basal and stimulated corticosterone output. However, this concentration of ouabain also inhibited steroidogenesis in fasciculata cells. 6. These results demonstrate a variety of situations where changes in intracellular [K+] are dissociated from those in corticosterone output and indicate that intracellular [K+] cannot be the sole mechanism regulating steroidogenesis under these conditions.

Effect of ACTH or zinc treatment on plasma aldosterone and corticosterone levels and on the in vitro steroid output from adrenocortical cells

1982 ◽  
Vol 60 (11) ◽  
pp. 1058-1064 ◽  
Author(s):  
Nicole Payet ◽  
Jean-Guy Lehoux
Keyword(s):  
Day Treatment ◽  
Zona Glomerulosa ◽  
Plasma Aldosterone ◽  
Female Rats ◽  
Zinc Hydroxide ◽  
Elevated Plasma ◽  
Adrenocortical Cells ◽  
Glomerulosa Cells ◽  
Zona Glomerulosa Cells

We have studied the effect of in vivo treatment with two forms of ACTH (Synacthen and Duracton) and of zinc hydroxide on plasma corticosteroid levels from adult Long Evans female rats. The corticosteroid output by isolated zona glomerulosa cells in vitro was also studied. Dose–response experiments showed that, after 2 days of treatment, Synacthen caused a 2.5- and 25.9-fold increase in plasma aldosterone and corticosterone levels, respectively, while maximal increases of 1.7- and 5.6-fold were obtained following treatment with Duracton. In contrast to elevated plasma steroid levels, the basal aldosterone and corticosterone output by isolated zona glomerulosa cells was significantly decreased for all doses of Synacthen administered. The 8 IU/day treatment with Synacthen produced an 86% diminution of aldosterone output while a treatment of 32 IU/day with Duracton gave only a 48.8% decrease. Concomitantly the Synacthen treatment provoked a high mitotic response in the zona glomerulosa cells (fivefold over control). A 2-week treatment with Synacthen resulted in elevated plasma aldosterone and corticosterone levels and produced a 92% diminution of aldosterone output by isolated zona glomerulosa cells. The aldosterone and corticosterone output from these cells was not enhanced by the addition of ACTH in the incubation media; zona glomerulosa cells of animals treated with Synacthen were no longer responsive to ACTH stimulation in vitro. The effect of a 2-day treatment with zinc hydroxide on plasma aldosterone and corticosterone levels and on steroid output by isolated adrenocortical cells was different from that of Synacthen. This meant that zinc was not the active principle of the Synacthen preparation. Our results indicate that long-term treatment with ACTH provokes profound functional changes at the adrenocortical zona glomerulosa level.

Biosynthetic and morphological evidence for inhibition of aldosterone production following administration of ACTH to sheep

1980 ◽  
Vol 94 (4) ◽  
pp. 559-570 ◽  
Author(s):  
John G. McDougal ◽  
Aldona Butkus ◽  
John P. Coghlan ◽  
Derek A. Denton ◽  
Jürg Müller ◽  
...  
Keyword(s):  
Time Course ◽  
Cell Number ◽  
Zona Glomerulosa ◽  
Morphological Evidence ◽  
Zona Fasciculata ◽  
Aldosterone Production ◽  
Glomerulosa Cells ◽  
17 Hydroxyprogesterone ◽  
Zona Glomerulosa Cells

Abstract. The effect of ACTH administration for 1—5 days on the morphology and steroidogenic capability of sheep adrenal tissue has been examined. During this period of treatment there was a gradual decline in the in vitro conversion of 3H-labelled precursors to products of solely zona glomerulosa origin (aldosterone and 18-hydroxycorticosterone) while conversion to products of zona fasciculata origin (17-hydroxyprogesterone, 11-deoxycortisol and cortisol) was stimulated throughout. Conversion to DOC, 18-hydroxydeoxycorticosterone and corticosterone (steroids produced by both the zona glomerulosa and the zona fasciculata) declined after initial stimulation. Within 2—3 days of the commencement of treatment, the zona glomerulosa showed a progressive decrease in cell number associated with disruption of cords and cell separation. Ultrastructurally, it was found that typical zona glomerulosa cells had almost disappeared. The majority of residual cells in this area had a structure intermediate between zona glomerulosa and zona fasciculata cells. The similarity in time-course of the alterations in both the morphological and biosynthetic characteristics suggests that the decline in aldosterone output caused by ACTH administration to sheep results from the loss of adrenal zona glomerulosa cells, predominantly due to selective cellular degeneration.

The properties of adrenal zona glomerulosa cells after purification by gravitational sedimentation

1974 ◽  
Vol 185 (1081) ◽  
pp. 375-407 ◽  
Keyword(s):  
Angiotensin Ii ◽  
Cyclic Amp ◽  
Microscopic Examination ◽  
Zona Glomerulosa ◽  
Maximal Response ◽  
Zona Fasciculata ◽  
Gravitational Sedimentation ◽  
Rat Adrenals ◽  
Glomerulosa Cells ◽  
Zona Glomerulosa Cells

The densities of latex spheres and biological cells can be reliably determined from their sedimentation rate in an albumin gradient under unit gravitational force. The densities of zona glomerulosa and fasciculata cells of rat adrenals were found to be 1.072 ± 0.004 and 1.040 ± 0.001 respectively. Purified zona glomerulosa cells of rat adrenals can be prepared by gravitational sedimentation of dispersed cells from capsule strippings of the gland, which originally contain 3 to10% zona fasciculata contamination. Electron and phase microscopic examination of the sedimented glomerulosa cells and their steroidogenic response to ACTH and cyclic AMP indicate that they are reasonably free of contamination from zona fasciculata cells. Electron microscopic examination of the purified glomerulosa cells indicates that most of them are reasonably normal in structure. Their basal production of corticosterone is decreased after sedimentation. However, their maximal response of corticosterone output to serotonin and potassium and their response to all potassium concentrations is not significantly altered, indicating normal function for the cells producing steroids. Their maximal responses to ACTH, valine angiotensin II and cyclic AMP are decreased, but, at the doses used, steroidogenesis by the zona fasciculata contamination in the unfractionated preparation would be stimulated by these substances. Purified zona glomerulosa cells have about the same maximal response of corticosterone output (about twofold) to potassium, valine and isoleucine angiotensin II, serotonin and ACTH. The maximal response of the purified zona glomerulosa cells to cyclic AMP is similar to that elicited by valine and isoleucine angiotensin II, potassium, serotonin or ACTH. This indicates that if these stimuli act by increasing cyclic AMP output, then the maximal response of corticosterone output (about twofold) is defined by the limited response of the biosynthetic pathways to cyclic AMP.

Inhibition of aldosterone release by hypoxia in vitro: interaction with carbon monoxide

1994 ◽  
Vol 76 (2) ◽  
pp. 689-693 ◽  
Author(s):  
H. Raff ◽  
B. Jankowski
Keyword(s):  
Carbon Monoxide ◽  
Angiotensin Ii ◽  
Zona Glomerulosa ◽  
Michaelis Constant ◽  
Aldosterone Synthase ◽  
Aldosterone Production ◽  
Glomerulosa Cells ◽  
In Vitro Interaction ◽  
Zona Glomerulosa Cells

We have demonstrated that the aldosteronogenic pathway of the zona glomerulosa is unusually sensitive to modest changes in PO2 (Michaelis constant for O2 approximately 95 Torr). The current study evaluated the interaction of CO (the classic ligand for P-450 enzymes) and the decreases in O2 on aldosteronogenesis in vitro. Bovine adrenocortical zona glomerulosa cells were incubated for 2 h and stimulated with either adenosine 3′,5′-cyclic monophosphate (cAMP) or angiotensin II. Ten and 20% CO led to significant decreases in cAMP- and angiotensin II-stimulated aldosteronogenesis. The combination of 20% CO and moderate decreases in PO2 (from approximately 140 to approximately 100 Torr) led to an interactive decrease in aldosterone production. The conversion of corticosterone to aldosterone catalyzed by aldosterone synthase, which is the site of O2 sensitivity, was not significantly inhibited by CO. We conclude that the aldosterone pathway is not exceptionally sensitive to CO compared with other steroidogenic pathways. This observation suggests that the unique O2-sensitive properties of the aldosterone pathway located primarily within aldosterone synthase may not reside in its CO binding site (i.e., heme).

Long-term effects of ACTH combined with angiotensin II on steroidogenesis and adrenal zona glomerulosa morphology in the rat

1987 ◽  
Vol 114 (1) ◽  
pp. 47-54 ◽  
Author(s):  
Anne M. Riondel ◽  
Piera Rebuffat ◽  
Giuseppina Mazzochi ◽  
Gastone G. Nussdorfer ◽  
Rolf C. Gaillard ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Morphological Changes ◽  
Zona Glomerulosa ◽  
Zona Fasciculata ◽  
Homogeneous Population ◽  
Long Term Treatment ◽  
Acth Treatment ◽  
Glomerulosa Cells ◽  
Zona Glomerulosa Cells

Abstract. To test the hypothesis that the trophic action of angiotensin II on the adrenal zona glomerulosa may allow a sustained stimulation of aldosterone by ACTH by preventing the morphological changes of the zona glomerulosa cells into zona fasciculata-like elements we investigated the effects in rats of a 6-day treatment with ACTH (100 μg/kg/day) alone or combined with angiotensin II (300 ng/kg/day) on corticosterone and aldosterone production and adrenal morphology. The responsiveness of both steroids to an acute ACTH dose was also studied on the last day of long-term treatment. Morphologic data showed that prolonged ACTH treatment stimulated the growth of zona glomerulosa cells, though it transformed the tubulo-lamellar cristae of mitochondria into a homogeneous population of vesicles. Angiotensin II furthered the trophic effects of ACTH but prevented the mitochondrial transformation. Despite its ability to conserve the well differentiated aspect of the zona glomerulosa cells, the administration of angiotensin II was unable to prevent the fall in the secretion of aldosterone caused by chronic ACTH treatment and its subsequent unresponsiveness to ACTH stimulation.

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