Effect of composition of incubation medium on aldosterone and corticosterone production by isolated rat zona glomerulosa cells

1982 ◽  
Vol 94 (2) ◽  
pp. 211-224 ◽  
Author(s):  
D. J. Campbell
Keyword(s):  
Angiotensin Ii ◽  
Cyclic Amp ◽  
Incubation Medium ◽  
Zona Glomerulosa ◽  
Bicarbonate Buffer ◽  
Angiotensin Ii Antagonist ◽  
Aldosterone Production ◽  
Glomerulosa Cells ◽  
Zona Glomerulosa Cells

The role of the composition of the incubation medium in determining the steroidogenic responsiveness of collagenase-dispersed rat zona glomerulosa cells was examined by studying the effect on production of aldosterone and corticosterone of (1) changes in the bovine serum albumin (BSA) concentration in Krebs–Ringer bicarbonate buffer (KRBGA), (2) dialysis of the BSA and (3) comparison of KRBGA with 'modified' Medium 199. Medium 199 was modified so that its electrolytic content was identical to that of KRBGA. Compared with 0·1–0·2% BSA in KRBGA, BSA concentrations of 0·5 and 4% caused inhibition of both basal and K+-stimulated, but not angiotensin II-stimulated steroidogenesis. This inhibitory property of BSA was not removed by dialysis. The BSA did, however, contain a dialysable factor which increased both basal steroidogenesis and the steroidogenic response to maximal K+ and angiotensin II stimulation. Both incubation media contained 0·2% BSA for the comparison of KRBGA with modified Medium 199. Modified Medium 199 increased both basal steroidogenesis and the aldosterone response to K+ stimulation (per cent increase above basal) by two- to threefold compared with KRBGA, with smaller increases in the response to ACTH and 5-hydroxytryptamine (5-HT) and a decrease in the response to cyclic AMP. In contrast, modified Medium 199 increased the aldosterone response to angiotensin II by sevenfold, from 60% (in KRBGA) to 420%. In KRBGA, angiotensin II inhibited K+-stimulated aldosterone production. This effect was produced by concentrations of angiotensin II below the threshold for steroidogenesis and could be reproduced with the angiotensin II antagonist [Sar1, Ileu8]-angiotensin II. Angiotensin II did not inhibit K+-stimulated aldosterone production in modified Medium 199. These data emphasize the importance of the composition of the incubation medium in determining the steroidogenic responsiveness of rat zona glomerulosa cells in vitro. Furthermore, these data indicate that the steroidogenic response to angiotensin II, compared with K+, ACTH, 5-HT and cyclic AMP, is more readily influenced by other, as yet unidentified, factors in the incubation medium, and are consistent with recent evidence that angiotensin II and K+ do not share a common mode of action on steroidogenesis by these cells.

Increased steroidogenesis by rat zona glomerulosa cells with increased cell concentration in vitro: evidence for a novel aldosterone-stimulating factor and implications regarding aldosterone biosynthesis

1982 ◽  
Vol 94 (2) ◽  
pp. 225-241 ◽  
Author(s):  
D. J. Campbell
Keyword(s):  
Incubation Medium ◽  
Cell Concentration ◽  
Zona Glomerulosa ◽  
Bicarbonate Buffer ◽  
Fourfold Increase ◽  
Aldosterone Production ◽  
Glomerulosa Cells ◽  
Stimulating Factor ◽  
Zona Glomerulosa Cells

A fourfold increase in the concentration of rat zona glomerulosa cells incubated in Krebs–Ringer bicarbonate buffer (KRBGA) increased aldosterone production per cell twofold. This cell concentration associated (CCA) increase in aldosterone production showed a dose–response relationship with the number of cells per incubation and was further increased in modified Medium 199, compared with KRBGA. The combination of a fourfold increase in cell concentration with each of maximally steroidogenic concentrations of K+, angiotensin II, ACTH, 5-hydroxytryptamine or cyclic AMP produced an increase in aldosterone production greater than that predicted from the algebraic sum of the steroidogenic effects of the factors acting separately. The CCA increase in basal aldosterone production was maximal during the first 30 min of incubation, could not be accounted for by accumulation of corticosterone in the incubation medium and was largely or completely abolished by the addition of pregnenolone, progesterone, deoxycorticosterone or corticosterone to the incubation medium. Together, these data support the proposal that the CCA effect was due to a novel aldosterone-stimulating factor which increased aldosterone production by stimulating pregnenolone formation. The aldosterone and corticosterone responses to exogenous pregnenolone, progesterone, deoxycorticosterone and corticosterone demonstrated that aldosterone and corticosterone production by zona glomerulosa cells in vitro may represent separate biosynthetic pathways. Furthermore, these studies provided evidence for a mechanism by which increased levels of exogenous pregnenolone, progesterone and deoxycorticosterone were able to increase their per cent conversion to aldosterone, but not to corticosterone.

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).

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.

Roles of type I and type II isoenzymes of cyclic AMP-dependent protein kinase in steroidogenesis in rat adrenal cells

1994 ◽  
Vol 12 (2) ◽  
pp. 195-202 ◽  
Author(s):  
B J Whitehouse ◽  
D R E Abayasekara
Keyword(s):  
Cyclic Amp ◽  
Cell Function ◽  
Zona Glomerulosa ◽  
Zona Fasciculata ◽  
Type I ◽  
Type Ii ◽  
Aldosterone Production ◽  
Dependent Protein ◽  
Glomerulosa Cells ◽  
Zona Glomerulosa Cells

ABSTRACT The role played by cyclic AMP (cAMP)-dependent protein kinases (PKAs) in rat adrenal steroidogenesis has been investigated using cAMP analogues which show partial selectivity for the type I and type II PKA isoenzymes. These were aminohexylamino-cAMP (AHA-cAMP; selective for site 1 on type I PKA), N6-benzoyl-cAMP (BZ-cAMP; selective for site 2 on PKA types I and II) and 8-thiomethyl-cAMP (TM-cAMP; selective for site 1 on type II PKA). Positive cooperativity exists between the two nucleotide-binding sites, thus the presence of type I PKA was inferred when synergistic increases in corticosteroid production were obtained with AHA-cAMP plus BZ-cAMP and that of type II PKA when synergistic increases were obtained with TM-cAMP plus BZ-cAMP. The effects of AHA-cAMP, TM-cAMP and BZ-cAMP (10–100 μmol/l) on aldosterone production by glomerulosa cell preparations and corticosterone production by fasciculata/reticularis cell preparations were compared. Dose-related stimulation of steroid production was obtained with each cAMP analogue in both types of cell preparation. Experiments were performed using the cAMP analogues in combination at doses which gave minimal stimulation individually. Cells were incubated with AHA-cAMP (66 and 100 μmol/l) or TM-cAMP (15, 30 and 45 μmol/l) in the presence and absence of 15μmol BZ-cAMP/l. Synergistic responses were obtained with both analogue pairs in both cell types. The synergism ratio in fasciculata/reticularis cell preparations for the type I PKA selective pair of analogues (100 μmol AHA-cAMP/l plus 15μmol BZ-cAMP/l) was significantly higher (P<0·01) than that for the type II selective pair (45μmol TM-cAMP/l plus 15μmol BZ-cAMP/l; 7·9±1·2 (mean±s.e.m.) and 2·6±0·3 respectively). In zona glomerulosa preparations the ratio was higher (P<0·05) for the type II selective pair (1·6±0·1 for AHA-cAMP plus BZ-cAMP and 2·8±0·4 for TM-cAMP plus BZ-cAMP). The effects of 100μmol AHA-cAMP/l and 45μmol TM-cAMP/l on the response to ACTH (1 pmol/l–10 nmol/l) were examined. Synergistic responses were obtained in fasciculata/reticularis cells with both analogues in combination with low concentrations of ACTH (10 and 100 pmol/l). In zona glomerulosa cells only the addition of TM-cAMP (45 μmol/l) in combination with 10 pmol ACTH/1 gave rise to synergistic increases in aldosterone production, which suggests that there may be some compartmentalization of the cAMP-dependent pathway in these cells. The results indicate that both isoenzymes of PKA are present in rat adrenocortical cells and can play a part in the control of steroidogenesis. Type I PKA activity appears dominant in the control of zona fasciculata/reticularis cell function whereas modulation of type II PKA activity plays a more significant role in the responses of zona glomerulosa cells.

Effects of Angiotensin II, Adrenocorticotropin, and Potassium on Aldosterone Production in Adrenal Zona Glomerulosa Cells from Streptozotocin-Induced Diabetic Rats*

Endocrinology ◽  
1986 ◽  
Vol 118 (1) ◽  
pp. 183-188 ◽  
Author(s):  
TOSHIKAZU KIGOSHI ◽  
NORIKO IMAIZUMI ◽  
SADAHIDE AZUKIZAWA ◽  
IKUO YAMAMOTO ◽  
KENZO UCHIDA ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Zona Glomerulosa ◽  
Diabetic Rats ◽  
Aldosterone Production ◽  
Glomerulosa Cells ◽  
Zona Glomerulosa Cells

LACK OF EFFECT OF ANGIOTENSIN ON LEVELS OF CYCLIC AMP IN ISOLATED ADRENAL ZONA GLOMERULOSA CELLS FROM THE RAT

1981 ◽  
Vol 91 (1) ◽  
pp. 145-154 ◽  
Author(s):  
J. B. G. BELL ◽  
J. F. TAIT ◽  
S. A. S. TAIT ◽  
G. D. BARNES ◽  
B. L. BROWN
Keyword(s):  
Angiotensin Ii ◽  
Bovine Serum Albumin ◽  
Cyclic Amp ◽  
Phosphodiesterase Inhibitor ◽  
Zona Glomerulosa ◽  
Zona Fasciculata ◽  
Angiotensin Iii ◽  
Independent Mechanism ◽  
Glomerulosa Cells ◽  
Zona Glomerulosa Cells

The effects of pure [Asp1, Val5]- and [Asn1, Val5]-angiotensin II and also [des-Asp1, Ile5]-angiotensin II (angiotensin III) on cyclic AMP and steroid outputs by dispersed rat capsular cells, comprising 95% zona glomerulosa and 5% zona fasciculata cells, have been studied. The results showed that [Asp1, Val5]- and [Asn1, Val5]-angiotensin II, at doses between 2·5 × 10−1 1 and 2 × 10−4 mol/l, which produced typical increases in steroidogenesis, failed to increase output of cyclic AMP. This lack of effect was observed whether the nucleotide was measured by radioimmunoassay or by adrenal binding protein and under the same conditions in which 8·4 mm-K+ consistently increased the output of cyclic AMP. Instead the results showed a small but significant decrease in cyclic AMP output with angiotensin II. Similar results were obtained with incubations for 60 rather than 120 min and with medium containing a concentration of 5 or 40 g bovine serum albumin/l. Although the levels of cyclic AMP were generally higher in the presence of the phosphodiesterase inhibitor, 3-isobutyl-l-methylxanthine, the same decrease relative to basal outputs was observed with angiotensin II which increased steroidogenesis. Angiotensin III also failed to increase output of cyclic AMP at doses (2·5×10−9 to 2·5×10−6 mol/l) which produced increases in steroid output equivalent to those obtained with angiotensin II. These results indicate that angiotensin II and III can act through a cyclic AMP- independent mechanism.

Effect of angiotensin II on aldosterone and its precursor steroid production in adrenal zona glomerulosa cells from heparin-treated rats

1986 ◽  
Vol 111 (2) ◽  
pp. 222-227 ◽  
Author(s):  
K. Uchida ◽  
S. Azukizawa ◽  
N. Imaizumi ◽  
T. Kigoshi ◽  
I. Yamamoto ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Corticosterone Level ◽  
Plasma Renin ◽  
Plasma Corticosterone ◽  
Zona Glomerulosa ◽  
Threshold Dose ◽  
Lower Response ◽  
Aldosterone Production ◽  
Glomerulosa Cells ◽  
Zona Glomerulosa Cells

Abstract. To assess the nature of the heparin-induced aldosterone deficiency, we investigated the stimulatory effect of angiotensin II (All) on aldosterone and its precursor steroids in adrenal zona glomerulosa cells from heparin-treated rats compared with those in the cells from vehicle-treated rats. Heparin-treated rats had low plasma aldosterone levels, high plasma renin activity and plasma All levels, and normal plasma corticosterone level 6 weeks after the treatment (1500 IU/kg, twice daily). Basal aldosterone production, when corrected to a uniform number of cells per group, was similar in the cells from heparin- and vehicle-treated rats. The cells from heparin-treated rats had a less sensitive and lower response of aldosterone production to All; an increase by 4 orders of magnitude in the threshold dose for All and a decrease in the maximum All-stimulated level. The maximum All-stimulated levels, but not the basal levels, of pregnenolone, corticosterone and 18-OHB production were low in the cells from heparin-treated rats. ACTH caused a similar stimulatory effect on aldosterone production in the cells from heparin- and vehicle-treated rats. The cells from heparin-treated rats had a less sensitive and lower response of aldosterone production to potassium; an increase by one order of magnitude in the threshold dose for potassium and a decrease in the maximum potassium-stimulated level, presumably because of the glomerulosa hyporesponsivness to AII. These results suggest that our heparin-treated rats have selective impairment of adrenal zona glomerulosa cells,

Effects of α-human atrial natriuretic polypeptide, sodium nitroprusside and dibutyryl cyclic GMP on aldosterone production in bovine zona glomerulosa cells

1988 ◽  
Vol 119 (3) ◽  
pp. 358-366 ◽  
Author(s):  
Mitsuhiro Okamoto
Keyword(s):  
Angiotensin Ii ◽  
Sodium Nitroprusside ◽  
Cyclic Gmp ◽  
Zona Glomerulosa ◽  
Atrial Natriuretic Polypeptide ◽  
Aldosterone Production ◽  
Cast Doubt ◽  
Glomerulosa Cells ◽  
Zona Glomerulosa Cells ◽  
Atrial Natriuretic

Abstract. When bovine adrenal zona glomerulosa cells were incubated with α-human atrial natriuretic polypeptide (α-hANP), the basal aldosterone production in the cells was hardly affected, although the angiotensin II- or K+-stimulated production was completely inhibited. α-hANP was found to cause the generation of cyclic GMP in the cells. When the cells were incubated with sodium nitroprusside, the drug inhibited the angiotensin II- or K+-stimulated aldosterone production, and also generated cyclic GMP in the cells. In contrast, dibutyryl cyclic GMP was found to be a stimulator of the aldosterone response rather than an inhibitor. The results obtained in this study cast doubt on the role of cyclic GMP as an intracellular second messenger for the action of ANP on aldosterone secretion.

Effect of exposure to hypoxia from birth on aldosterone in rabbits: role of unesterified fatty acids

1997 ◽  
Vol 272 (4) ◽  
pp. R1084-R1087 ◽  
Author(s):  
H. Raff ◽  
B. M. Jankowski ◽  
T. L. Goodfriend ◽  
J. E. Baker ◽  
P. E. Papanek
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Zona Glomerulosa ◽  
Hypoxic Hypoxia ◽  
Adrenocortical Function ◽  
Total Serum ◽  
Aldosterone Production ◽  
Glomerulosa Cells ◽  
Zona Glomerulosa Cells

Hypoxia and fluid and electrolyte disturbances are serious risks to normal postnatal development. Because a decrease in inspired O2 (hypoxic hypoxia) inhibits aldosterone synthesis in the adult and aldosterone controls water and electrolyte balance, we studied adrenocortical function in rabbits exposed to normobaric normoxia or hypoxic hypoxia (fraction of inspired O2 0.09) from birth. At 21 days of age, rabbits were anesthetized, the adrenals were rapidly removed, and the adrenal capsules containing mostly zona glomerulosa cells were separated. Cells were dispersed with collagenase and studied in vitro. Hypoxia in vivo resulted in a 73% decrease in basal aldosterone release and a 86% decrease in adenosine 3',5'-cyclic monophosphate-stimulated aldosterone release in vitro. We hypothesized that increased unesterified fatty acids could be partly responsible for inhibition of aldosterone synthesis. Total serum unesterified fatty acids in hypoxic kits were significantly increased (298 +/- 14 micromol/l) compared with normoxic kits (184 +/- 31 micromol/l). When cells from hypoxic rabbits were washed with fatty acid-free albumin and studied under conditions devoid of fatty acids, aldosterone production was partially restored. Corticosterone production was not affected by washing. Washing had no effect on aldosterone synthesis by cells from normoxic rats. Finally, exposing washed zona glomerulosa cells to oleic acid (10-50 microM) inhibited aldosteronogenesis. We conclude that exposure to hypoxia from birth attenuates aldosterone production in part due to an increase in levels of unesterified fatty acid levels.

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