Stimulation of steroidogenesis by forskolin in rat adrenal zona glomerulosa cell preparations

1991 ◽  
Vol 129 (3) ◽  
pp. 391-397 ◽  
Author(s):  
S. J. Purdy ◽  
B. J. Whitehouse ◽  
D. R. E. Abayasekara
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Zona Glomerulosa ◽  
Zona Fasciculata ◽  
Low Doses ◽  
Significant Stimulation ◽  
Glomerulosa Cells ◽  
High Doses ◽  
Zona Glomerulosa Cells ◽  
Stimulation Of

ABSTRACT The actions of forskolin have been investigated to determine to what extent its effects on steroidogenesis in rat adrenal preparations are dependent on activation of adenylate cyclase. In zona glomerulosa preparations, stimulation of both aldosterone and corticosterone production was obtained at concentrations of forskolin between 1 and 10 μmol/l. The effects of 10 μmol forskolin/l were additive with those of low doses (1 pmol/l) of corticotrophin (ACTH), but not with those of high doses (1 nmol/l) of ACTH. In contrast, in zona fasciculata/reticularis cells, doses of forskolin up to 10 μmol/l produced no significant stimulation of corticosterone production either alone or in the presence of ACTH (1 pmol/l and 1 nmol/l). The response to 1 nmol ACTH/l was attenuated in the presence of forskolin (10 μmol/l) in both zona glomerulosa and zona fasciculata/reticularis cell preparations. Cyclic AMP production increased progressively with dose up to 100 μmol forskolin/l in zona glomerulosa cells, whereas corticosterone production was maximal between 10 and 30 μmol forskolin/l and decreased at 100 μmol forskolin/l. In zona fasciculata/reticularis cells, cyclic AMP production was also increased by forskolin (1 and 10 μmol/l). The stimulation of zona glomerulosa steroido-genesis by forskolin (1–10 μmol/l) and ACTH (1–100 pmol/l) were both reduced by the adenylate cyclase inhibitor, N6-phenylisopropyladenosine (100 μmol/l). The calcium channel inhibitor, nifedipine, only reduced the steroidogenic response to forskolin (3 μmol/l) at doses of 300 μmol/l whereas the response to 8·4 mmol K+/l was inhibited at 10 μmol nifedipine/1. Although there is some dissociation between the effects of forskolin on cyclic AMP and steroidogenesis, the results are generally consistent with the view that the effects of forskolin in rat zona glomerulosa cells are mainly dependent on activation of adenylate cyclase. This contrasts with the effects of forskolin in bovine fasciculata cells which are reported to be mediated by activation of voltage-regulated calcium channels. Journal of Endocrinology (1991) 129, 391–397

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.

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

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.

The mechanism of the effect of K + on the steroidogenesis of rat zona glomerulosa cells of the adrenal cortex: role of cyclic AMP

1986 ◽  
Vol 227 (1246) ◽  
pp. 21-42 ◽  
Keyword(s):  
Cyclic Amp ◽  
Adrenal Cortex ◽  
Zona Glomerulosa ◽  
Free Concentration ◽  
Mean Values ◽  
Glomerulosa Cells ◽  
Zona Glomerulosa Cells ◽  
Stimulation Of

The effects of various concentrations of extracellular K + (3.6 - 13 mM) on the steroid (corticosterone and aldosterone) and cyclic AMP outputs of capsular cells (95% zona glomerulosa) of the rat adrenal cortex were studied at different concentrations of extracellular Ca 2+ . Small amounts of EGTA (50 μM) were added to reduce the free Ca 2+ concentrations effectively to zero at the lowest possible total Ca 2+ concentration. At a total extracellular concentration of 2.5 mM Ca 2+ , in 27 experiments the mean values of the steroid and cAMP outputs showed a maximum at 8.4 mM K + . The increase in steroid and cAMP outputs at 5.9, 8.4 and 13 mM K + compared with that at 3.6 mM were highly significant ( p < 0.01). The overall correlation of either corticosterone or aldosterone with cAMP outputs was also highly significant and was even better from 3.6 to 8.4 mM K + . Lowering the effective free concentration of Ca 2+ to zero decreased the steroid and cAMP outputs significantly at all K + concentrations, and no output was then significantly higher than at 3.6 mM. With the pooled data on outputs at all total Ca 2+ (2.5, 0.5, 0.25, 0.10, 0.05 and 0.0 mM) and K + (3.6, 5.9, 8.4 and 13 mM) concentrations, the correlation of either steroid with cAMP outputs was highly significant (but again optimally from 3.6 to 8.4 mM K + ). Nifedipine (10 -6 to 10 -4 M) was added to the incubations with the aim of specifically inhibiting Ca 2+ influx at total extracellular Ca 2+ concentra­tions of 2.5, 1.25 and 0.25 mM and with the usual K + concentrations. The cAMP outputs were reduced at all K + concentrations above 3.6 mM K + . The effect was highly significant at 10 -4 M nifedipine and a total Ca 2+ of 1.25 mM, which with the incubation conditions used, corresponds to the free Ca 2+ concentrations in vivo . These results indicate that cAMP plays a significant role in the stimulation of steroid output by K + particularly between 3.6 and 8.4 mM K + . In this range of K + concentrations the stimulation of cAMP seems to be controlled by increases in Ca 2+ influx. The correlation of steroid and cAMP output at the higher K + concentra­tions (between 8.4 and 13 mM K) and at the various total Ca 2+ concentra­tions is less significant. Also, with all concentrations of added nifedipine there is an ‘anomalous’ increase in steroid output at 13 mM K + and at total Ca 2+ concentrations of 2.5 and 1.25 mM. However, at the same K + concentrations and at 0.25 mM Ca 2+ , nifedipine decreases steroid outputs. Our previous data, obtained after addition of maximally effective amounts of cAMP, indicated that there were also non-cAMP mechanisms involved in the stimulation of steroidogenesis by K + in z. g. cells. The present data confirm this conclusion, particularly at K + concentrations above 8.4 mM. They also indicate that at these higher K + concentrations, by non-cAMP mechanisms increasing intracellular Ca 2+ concentrations probably inhibit steroidogenesis. We conclude, however, that in the physiological range of K + concentra­tions, the role of cAMP in zona glomerulosa cells is at least comparable in importance to that of non-cAMP mechanisms.

Effects of α-melanocyte-stimulating hormone on the cyclic AMP and phospholipid metabolism of rat adrenocortical cells

1986 ◽  
Vol 110 (3) ◽  
pp. 405-416 ◽  
Author(s):  
P. J. Hyatt ◽  
J. B. G. Bell ◽  
K. Bhatt ◽  
F. W. Chu ◽  
J. F. Tait ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Cyclic Amp ◽  
Phosphatidic Acid ◽  
Inositol Phosphates ◽  
Phospholipid Metabolism ◽  
Zona Glomerulosa ◽  
Zona Fasciculata ◽  
Additional Increase ◽  
Glomerulosa Cells ◽  
Stimulation Of

ABSTRACT Results on the effects of peptides on the phospholipid metabolism and steroid and cyclic AMP (cAMP) outputs of rat adrenal capsular cells (96% zona glomerulosa, 4% zona fasciculata) were obtained in a series of three batch experiments. Their significance was examined by analysis of variance. Incorporation of [32P] into phosphatidylcholine, phosphatidic acid and phosphatidylinositol was measured. Production of [3H]inositol-1 monophosphate, inositol-1,4 bis-phosphate and inositol-1,4,5 tris-phosphate was estimated after prelabelling with [3H]inositol followed by 1 min incubation with a steroidogenic stimulus. Angiotensin II (0·25 nmol/l to 0·25 μmol/l) highly significantly (P < 0·01) stimulated aldosterone and corticosterone outputs, [32P] incorporation into phosphatidic acid and phosphatidylinositol (but not into phosphatidylcholine) and the production of the three [3H]inositol phosphates. Aldosterone and corticosterone outputs were stimulated by α-MSH (above 0·1 nmol/l). However, incorporation of [32P] was not significantly increased until 10 μmol α-MSH/l but, unlike with angiotensin II, incorporation into phosphatidylcholine was also then stimulated. Also, the production of the inositol phosphates was not increased significantly (P > 0·05) by any dose of α-MSH (10 nmol/l, 1 μmol/l and 0·1 mmol/l) used. Therefore, it can be concluded that α-MSH does not stimulate phospholipase C in rat zona glomerulosa cells. In further experiments, it was also found that there were significant increases in cAMP as well as in steroid outputs above 1 nmol α-MSH/l (highly significant above 10 nmol α-MSH/l). There were plateaux of the outputs of both steroids and cAMP from 0·1 to 1 μmol α-MSH/l. However, there were further increases in steroid and cAMP outputs of the capsular cells at higher doses. Concomitant results on the stimulation of corticosterone output by zona fasciculata–reticularis cells indicate that this additional increase was mostly due to the stimulation of the contaminating zona fasciculata cells. It was also confirmed that α-MSH preferentially stimulates steroidogenesis by the zona glomerulosa. However, under our conditions, α-MSH highly significantly increased the output of cAMP by both zona fasciculata and glomerulosa cells. J. Endocr. (1986) 110, 405–416

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.

Sign in / Sign up

Export Citation Format

Share Document