Sylvia Agnes Sophia Tait

The Properties of Adrenal Zona Glomerulosa Cells after Purification by Gravitational Sedimentation

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.

The discovery, isolation and identification of aldosterone: reflections on emerging regulation and function

This paper has a focus on the early history of aldosterone. The Taits take us on a chronological trawl through the history in which they had a first hand role and made a major contribution-their bioassay was in many ways the key. The gifted Swiss chemists made a critical contribution to the scale and isolation of larger amounts. This was international collaboration at its best. Developing technologies were utilised as crucial cutting edge applications in the advancing front, technology transfer before the word was invented. Measurement of aldosterone and angiotensin were crucial advances to the understanding of the regulation of the hormone. In the period 1960-2003, some 30,000 papers mentioned aldosterone as a keyword, even so advances on a larger scale were slow. I have indicated some of my own work with the Howard Florey team using the adrenal autotransplant in the conscious sheep. Recently, the understanding of the role of induced proteins, the flow on from the RALES trial and the development of eplerenone has revitalised the aldosterone field.

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

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 Ca2+. Small amounts of EGTA (50 μM) were added to reduce the free Ca2+ concentrations effectively to zero at the lowest possible total Ca2+ concentration. At a total extracellular concentration of 2.5 mM Ca2+, 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 Ca2+ 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 Ca2+ (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 Ca2+ influx at total extracellular Ca2+ 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 Ca2+ of 1.25 mM, which with the incubation conditions used, corresponds to the free Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ concentrations of 2.5 and 1.25 mM. However, at the same K+ concentrations and at 0.25 mM Ca2+, 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 Ca2+ 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.

Purification of Dispersed Rat Adrenal Cells by Column Filtration

A simple and rapid method for the purification of specific cell populations in dispersed rat adrenal capsular and decapsulated preparations is described. Dispersed cell preparations were filtered by passage through a column of Sephadex G-10 (for decapsulated preparations) or G-15 (for capsular preparations). The smaller cells, i. e. zona reticularis in decapsulated preparations and zona glomerulosa in capsular preparations, appeared in the filtrate and were separated from the larger zona fasciculata cells, which were retained on the column in both situations. In six capsular preparations, contamination by zona fasciculata cells was reduced from 7.3 ± 1.1% (mean ± s. e.) to 0.28 ± 0.09%. The recovery of zona glomerulosa cells was 18.6 ± 2.2% of those applied to the column. Corticosterone output per million cells was reduced, after filtration, to 48% of the output by unpurified cells and aldosterone output was reduced to 10-20% of that of unpurified cells. When expressed as a stimulation ratio (S = stimulated output/basal output), the response of corticosterone output to 8.4 mm [K+] was not altered significantly after filtration. S values were 3.04 ± 0.26 and 2.27 ± 0.48 for unpurified and filtered cells respectively, but the S value for adrenocorticotrophic hormone (ACTH), which preferentially stimulated the contaminating zona fasciculata cells, decreased significantly from 6.40 ± 0.49 to 2.54 ± 0.44 (p < 0.001). Both the steroid outputs and S values were similar to those reported by Tait, Tait, Gould & Mee (Proc. R. Soc. Lond. B 185, 375 (1974)) for zona glomerulosa cells after purification by unit gravity sedimentation. Cells retained by the column and retrieved by subsequent resuspension and filtration to remove Sephadex particles showed steroidogenic characteristics similar to those of unpurified cells. Filtration of decapsulated preparations resulted in recoveries of 44.6 ± 3.0% of loaded reticularis cells and 4.7 ± 0.7% of loaded fasciculata cells after filtering 18 adrenal equivalents for 10 min (28 experiments). In four experiments with 36 adrenal equivalents filtered for 5 min, recoveries were 35.9 ± 5.1% and 1.5 ± 0.4% respectively. The retained cell preparation showed 50% enrichment of zona fasciculata cells. After filtration according to the former protocol, cells secreted more deoxycorticosterone (DOC) and less corticosterone per million cells than either the unpurified or retained cell preparations. The filtered cells were less responsive to stimulation by ACTH, the effect being most marked for corticosterone output, for which the S value decreased from 126 ± 13 for unpurified and 131 ± 4.5 for retained cells to 15.8 ± 5.2 for filtered cells. Corresponding S values for deoxycorticosterone were 13.9 ± 1.3, 22.7 ± 4.7 and 8.3 ± 3.0. Basal R (DOC output/corticosterone output) values of 0.41 ± 0.05, 0.25 ± 0.07 and 1.20 ± 0.32 for unpurified, retained and filtered cells respectively, were observed. Steroid outputs and S and R values for filtered and retained cells were, therefore, similar to those for the equivalent zona reticularis and zona fasciculata preparations, obtained by unity gravity sedimentation, by Bell, Gould, Hyatt, Tait & Tait (J. Endocr. 77, 9P (1978)). The major advantage of the column filtration procedure for preparation of specific cell populations is that it is rapid and simple in terms of both equipment and manpower, while maintaining a high degree of enrichment and acceptable cell recovery levels and yielding cells that are found by both morphological and functional criteria, to be viable.

The Use of the Superfusion Approach with Rat Adrenal Capsular Cells to Compare the Steroidogenic Potencies of Angiotensin Analogues, without the Effects of Peptide Degradation

The superfusion approach was used to assay the relative potencies of (Asp1)angiotensin II (A II), (des-Asp1)angiotensin II (A III) and (Sar1)angiotensin II (SA II) in stimulating the steroidogenesis (corticosterone) of rat adrenal capsular (zona glomerulosa) cells without the effects of peptide degradation; an example of the advantages of such a procedure. At the rate of superfusion employed, there was no significant reduction in the concentration of A ll or A III through the cell chamber. However, in the in vitro closed system A III was degraded more rapidly than A ll. SA II would be expected to be more protected than A ll. The potency ratios were found to be 0.30±0.05 (s.e.), four experiments for A III/A II and 2.23±0.16 (s.e.), six experiments for SA II/A II in the usual closed system with 1 h incubations. In the superfused system, however, the corresponding ratios were 0.82±0.11, five experiments and 1.19 ±0.23, four experiments. These results indicated that, as shown by the relative potencies in the superfusion system and therefore without the effects of degradation. A ll, A III and SA II have about the same intrinsic activity. The reason for the relatively high activity of SA II in the closed system is its lower rate of degradation compared with A ll and the low activity of A III is owing to its relatively high rate of degradation. It is concluded that, although the intrinsic activities of A III and A ll are similar, because of the equivalent activity of SA II, it is probably not necessary for SA II, or therefore also A ll, to be converted to A III for it to stimulate steroidogenesis in the system.