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I. M. Bird, B. C. Williams, and S. W. Walker


Bovine adrenocortical cells from the zona fasciculata/reticularis were isolated and their phosphoinositides labelled to a steady state with [3H]inositol in primary culture. Experiments performed on these cells in the presence of Li+ have shown that, over a period of 60min, angiotensin II (AII; 10−7 m) stimulated a linear increase in [3H] inositol phosphates that was sustained through the utilization of two hormone-sensitive subpools of prelabelled lipid (30% and 45% respectively), and a rapid resynthesis of [3H]phosphoinositide into one of these pools using cytosolic [3H]inositol. The 30% pool was used immediately on stimulation, and was sustained at a steady-state size of 10–15% during the first 30 min of stimulation through rapid resynthesis using cytosolic [3H]inositol. Only after 30min, when the cytosolic [3H]inositol was depleted and resynthesis could no longer occur, did the additional 45% pool start to supply further substrate to the phospholipase C, thereby further sustaining the generation of [3H]inositol phosphates. Once this pool was depleted however (by approximately 60min), [3H]inositol phosphate generation finally ceased. These findings establish the differential use of two metabolically distinct hormone-sensitive pools of phosphoinositide following AII stimulation in bovine adrenocortical cells, events which are dependent upon the availability of cytosolic inositol for phosphoinositide resynthesis.

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I. M. Bird, S. W. Walker, and B. C. Williams


In 1980, studies of the hormone regulation of adrenocortical steroidogenesis had reached a turning point. The important differences in function and responsiveness of the different adrenocortical zones had been recognized (Tait, Tait & Bell, 1980; see also Brown, 1982), and the need for purified cell populations from each zone for in-vitro studies emphasized. Two reviews of that year (Schimmer, 1980; Tait et al. 1980) also highlighted advances which had been made in understanding the mechanisms of hormone-stimulated (particularly adrenocorticotrophin (ACTH)-stimulated) cyclic AMP (cAMP) generation in the adrenal cortex, and how cAMP could bring about an increase in adrenal steroidogenesis. However, these reviews also stressed that not all the known steroidogenic agonists stimulated cAMP production. At least one agonist (angiotensin II (AII)) operated through a mechanism requiring an increase in intracellular Ca2+ concentration ([Ca2+]i).

In many other tissues, agonists such as AII, vasopressin and acetylcholine were known

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S. W. Walker, M. W. J. Strachan, M. Nicol, B. C. Williams, and I. M. Bird


The effects of angiotensin II (AII), acetylcholine and vasopressin on the intracellular concentration of Ca2+ have been little studied in adrenocortical cells from the zona fasciculata/reticularis (ZFR).

Primary cultures of bovine ZFR cells maintained in suspension culture for 72 h produce cortisol in response to AII (0·1 μm), acetylcholine (0·1 mm) and vasopressin (1 μm). This response is accompanied by a breakdown of membrane phosphoinositides from [3H]inositol-prelabelled cells.

Using cells loaded with the Ca2+ indicator fura-2, the intracellular concentration of Ca2+ was measured in response to increasing doses of all three agonists and found to increase in a graded fashion in each case. The basal intracellular concentration of Ca2+ was 75±3 nm (mean±s.e.m., n=52), rising to a maximum 1·82±0·14-fold (n=6) for AII (0·1 μm), 1·35±0·05-fold (n=7) for acetylcholine (0·1 mm) and 1·27±0·10-fold (n=6) for vasopressin (1 μm).

In the case of AII and acetylcholine, agonists were added sequentially in medium of normal extracellular Ca2+ concentration (1·2 mm) or in medium in which the Ca2+ concentration was buffered to approximate to the intracellular concentration of Ca2+ (75–100 nm). Evidence was thereby obtained that both AII and acetylcholine mobilize a common intracellular pool of Ca2+.

Our findings suggest that these three agonists, all of which stimulate phospholipase C, increase intracellular Ca2+ through a mechanism which depends, at least in part, on the release of Ca2+ from a common intracellular pool.

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N. Hanley, B. C. Williams, M. Nicol, I. M. Bird, and S. W. Walker


Using tritiated-thymidine incorporation as a measure of cell growth, interleukin-1β stimulated the growth of bovine zona fasciculata/reticularis adrenocortical cells after 72h in primary culture. Within the range of 10–1000pg/ml, interleukin-1β produced over 40% of angiotensin II-stimulated [3H]thymidine incorporation (P<0.005 compared with basal for 10pg/ml and 1000pg/ml; P<0.05 for 100pg/ml; two-tailed unpaired Student's t-test). Interleukin-1β did not directly stimulate cortisol secretion.

By stimulating adrenocortical growth, the increase in interleukin-1 during fever provides a potential mechanism for chronically raising glucocorticoid output. This study is the first demonstration of a long-term effect involving interleukin-1β on the adrenal cortex.

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I. M. Bird, M. Nicol, B. C. Williams, and S. W. Walker


Cells isolated from the zona fasciculata/reticularis (ZFR) of the bovine adrenal cortex and maintained in culture were found to secrete cortisol in response to vasopressin stimulation. The increased cortisol secretion was dose dependent, with a threshold response at 1 nm and a maximal response (1·68-fold over basal) at 0·1 μm. In cells cultured in the presence of [3H]inositol (to prelabel the membrane phosphoinositide pool), stimulation with vasopressin in the presence of LiCl (10 mm) resulted in a similar dose-dependent increase in labelling of the phosphoinositol fraction, with a maximal response (1·45-fold over basal) at 10 nm. The increased labelling of the phosphoinositol fraction was independent of extracellular Ca2+ as it was not abolished in medium with [Ca2+ ] buffered to intracellular resting levels. This suggests that vasopressin stimulation results in the activation of a phosphoinositidase C. It is probable that cortisol secretion by bovine ZFR cells in response to vasopressin is dependent upon activation of this Ca2+-independent phosphoinositidase C. However, the small magnitude of the cortisol secretory response makes it unlikely that vasopressin is a primary regulator of cortisol secretion in vivo.