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.

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.

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.

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.

Neuropeptide Y modulates the sensitivity of the rat adrenal cortex to stimulation by ACTH

1995 ◽  
Vol 145 (2) ◽  
pp. 283-289 ◽  
Author(s):  
J P Hinson ◽  
L A Cameron ◽  
S Kapas
Keyword(s):  
Angiotensin Ii ◽  
Neuropeptide Y ◽  
Adrenal Cortex ◽  
Mammalian Species ◽  
Zona Glomerulosa ◽  
Zona Fasciculata ◽  
Steroid Secretion ◽  
Glomerulosa Cells ◽  
Zona Glomerulosa Cells

Abstract Neuropeptide Y (NPY) has been identified in nerves supplying the adrenal cortex of several mammalian species, although its function in this tissue is unknown. The present studies, employing adrenocortical cells prepared by collagenase digestion, have shown that NPY, in the absence of other stimulants, has no effect on steroid secretion by the rat adrenal over a range of peptide concentrations (10−11 to 10 −6 mol/l). However, in the presence of physiological concentrations of ACTH, which are submaximal for the stimulation of aldosterone secretion, NPY (10−6 mol/l) significantly enhanced the secretion rate of aldosterone by rat zona glomerulosa cells in response to ACTH. This effect was specific to the rat zona glomerulosa as NPY had no effect on the response to ACTH in rat zona fasciculata cells. The effect of NPY appears to be biphasic, however, as NPY significantly attenuated the steroidogenic response to supramaximal ACTH concentrations: in rat zona glomerulosa cells the aldosterone response to 10 −8 mol ACTH/l was significantly inhibited by NPY. The effect of NPY on the ACTH response appeared to be mediated by changes in the cAMP response. NPY had no effect on the steroidogenic response to potassium ions (K+), but enhanced the response to angiotensin II. NPY (10 −6 mol/l) significantly stimulated inositol 1,4,5-trisphosphate (InsP3) production although this concentration of peptide had no effect on steroid secretion. The effects of NPY on InsP3 production were additive with those of angiotensin II. These results suggest that the role of NPY in the adrenal cortex may be to regulate the sensitivity of the zona glomerulosa to peptide stimulation. Journal of Endocrinology (1995) 145, 283–289

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.

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

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

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