ABSTRACT

Accumulated data from in-vitro experiments have suggested that 18-hydroxysteroids may be stored within the intact rat adrenal zona glomerulosa. The phenomenon was further investigated by comparing the amount of steroid remaining in the zona glomerulosa tissue with that secreted into the media during incubation in vitro. The results showed that 18-hydroxydeoxycorticosterone (18-OH-DOC) and 18-hydroxycorticosterone (18-OH-B) were retained within the tissue against a considerable concentration gradient, with smaller amounts of aldosterone and corticosterone. Lysis of the intact zona glomerulosa, by preincubation in distilled water, yielded an enriched plasma membrane preparation. After subsequent incubation in Krebs–Ringer bicarbonate this preparation contained significantly more 18-OH-DOC than did the intact tissue, suggesting that tissuesequestered 18-OH-DOC is normally metabolized to other products. These may include 18-OH-B and aldosterone.

Fractionation of homogenized intact zona glomerulosa and the enriched plasma membrane preparation by density gradient centrifugation showed that tissue 18-OH-DOC banded in fractions of density 1·063– 1·21 g/ml and that its distribution was highly correlated with protein. Corticosterone, 18-OH-B and aldosterone banded like added free [³H]18-OH-DOC in fractions of density < 1·006 g/ml.

The results suggest that 18-OH-DOC is the major sequestered steroid within the rat adrenal zona glomerulosa and that this sequestration is attributable to the association of 18-OH-DOC with a high-density component of the plasma membrane.

J. Endocr. (1988) 117, 191–196

ABSTRACT

The finding that the rat adrenal zona glomerulosa cell shows specific sensitivity to stimulation by α-MSH and related peptides has been confirmed both in vivo and in vitro, raising the possibility that α-MSH may have a physiological role in the control of glomerulosa function and aldosterone secretion. To define more closely the structural features which confer the specificity of the glomerulosa response, other ACTH derived peptides have been tested for their specificity of actions on rat adrenal cells in vitro. The peptides tested were ACTH(5–24), ACTH(1–12), ACTH(1–14), ACTH(1–15), ACTH1–16) and ACTH(1–17). Their actions were compared with those of α-MSH and ACTH(1–24). All of the ACTH-derived peptides stimulated glomerulosa corticosterone production with sensitivities similar to that of α-MSH; minimum effective concentration was 10 nmol/l. Also, like α-MSH, the shorter ACTH peptides stimulated aldosterone production only relatively weakly in these cells from animals on normal sodium intake. Only ACTH(5–24), ACTH(1–16) and ACTH(1–17) stimulated fasciculata/reticularis cells at concentrations up to 1 μmol/l. The actions of all of the shorter peptides were thus unlike those of ACTH(1–24) which stimulates both cell types with approximately equal sensitivity, and which furthermore strongly stimulates aldosterone production.

The data suggest that the 18–24 region of the ACTH molecule contains the signal for a fasciculata/ reticularis response, and the region 1–13 that for glomerulosa specificity. They confirm the view that, in the rat, α-MSH itself may be the specific pituitary glomerulosa-stimulating agent which much experimental work has predicted. They also indicate that synthetic ACTH(1–17) analogues should be used with caution.

J. Endocr. (1986) 109, 275–278

ABSTRACT

The responses of human adrenocortical cells to stimulation by ACTH(1–24), desacetyl-α-MSH, α-MSH and angiotensin II amide have been compared. Both desacetyl-α-MSH, thought to be the major form of the peptide in the human pituitary and in circulating plasma, and α-MSH caused a significant stimulation of aldosterone, corticosterone and cortisol secretion. Significant stimulation of the production of these steroids was obtained with desacetyl-α-MSH at a concentration of 1 nmol/l, while the response to α-MSH was considerably less sensitive, with a minimum effective concentration of 0·1 μmol/l. These values compared with minimum effective concentrations of 1 pmol/l for ACTH and 0·1 μmol/l for angiotensin II amide. Although cell types were not separated, it is possible to conclude that none of the peptides showed any specificity for the zona glomerulosa, and in each case the same minimum effective concentration of peptide was required for both aldosterone and cortisol secretion. Yields of steroid obtained under conditions of maximal stimulation by ACTH(1–24), α-MSH and desacetyl-α-MSH were at least three to five times the basal output of aldosterone, four to eight times that for corticosterone and more than seven to sixteen times that for cortisol. Angiotensin II amide was a relatively poor stimulant with maximal stimulation only 1·5 × basal. In these experiments the minimum effective concentration for desacetyl-α-MSH (1 nmol/l) was close to the circulating concentration of desacetyl-α-MSH (0·3 nmol/l) in man, and it is thus possible that this peptide may have a physiological role in the control of adrenocortical function.

Journal of Endocrinology (1989) 121, 579–583

ABSTRACT

The time-course for the in-vitro secretion of aldosterone and 18-hydroxycorticosterone (18-OH-B) by rat adrenal whole capsular tissue (largely zona glomerulosa) was studied under control and stimulated conditions. The stimulatory effect of trypsin was relatively delayed, and the steroids were significantly enhanced only after 1 h, in contrast to the actions of ACTH, which produced effects after 15 or 30 min. Tissue-sequestered 18-hydroxydeoxycorticosterone (t-18-OH-DOC), which is not affected by ACTH, was significantly depleted by trypsin, but secreted 18-OH-DOC was not consistently affected by either stimulant. In contrast to the apparent mobilization of t-18-OH-DOC, the conversion of exogenously added [³H]18-OH-DOC to [³H]18-OH-B was inhibited by trypsin, and aldosterone was unaffected. When trilostane was added to inhibit de-novo steroidogenesis, under conditions in which the steroid secretory response to ACTH is completely inhibited, aldosterone and 18-OH-B secretion was still stimulated by trypsin although yields were lower. Compared with controls, trilostane reduced t-18-OH-DOC concentrations, and trypsin caused a further depletion.

In other studies, glomerulosa plasma membrane enriched preparations were homogenized and centrifuged, and the supernatants were dialysed and added to incubations of dispersed zona glomerulosa cells in the presence or absence of stimulators of aldosterone secretion. The addition of the supernatants, which contained high concentrations of sequestered t-18-OH-DOC, stimulated aldosterone and 18-OH-B production by collagenase-dispersed zona glomerulosa cells to a greater extent than the addition of an equivalent amount of free 18-OH-DOC or corticosterone. When trypsin, ACTH, the phorbol ester phorbol myristate acetate or increased potassium were also added, there was a further increase in 18-OH-B production, and final recoveries of 18-OH-DOC were correspondingly decreased.

The results are consistent with the hypothesis that, because of the nature of its disposition in the glomerulosa cell, t-18-OH-DOC may be utilized as a substrate for aldosterone and 18-OH-B production. The plasma membrane location of this stored steroid pool, and the known actions of phorbol ester or trypsin stimulation, suggest that it may be mobilized by protein kinase C activation.

Journal of Endocrinology (1992) 135, 125–133

ABSTRACT

The role of the pituitary gonadotrophins in controlling luteal function in the stumptailed macaque has been investigated by examining profiles of serum concentrations of LH, FSH, progesterone and oestradiol in daily blood samples from 13 monkeys during the menstrual cycle, and in blood samples taken at hourly intervals between 09.00 and 21.00 h on different days of the luteal phase in 13 cycles. The effects of acute withdrawal of gonadotrophins was investigated by administering a single injection of 300 μg LHRH antagonist/kg body weight at different stages of the luteal phase during 28 cycles.

Although there were high basal values and marked fluctuations of bioactive LH during the first 4 days after the LH peak, progesterone profiles showed no corresponding short-term changes, there being a slow and steady rise in progesterone concentrations during the sampling periods. After day 5, basal LH secretion decreased, but high amplitude LH pulses were identified which were associated with episodes of progesterone secretion.

Administration of the LHRH antagonist caused a suppression of bioactive LH and progesterone concentrations at all stages of the luteal phase, although some basal secretion of progesterone was maintained through the 24-h period of effective antagonist gonadotroph blockade. Luteal function recovered apparently normally in all monkeys treated in the early–mid-luteal phase.

Serum concentrations of FSH and oestradiol fluctuated comparatively less during the 12-h sampling periods, and the antagonist had less suppressive effects on the concentrations of these hormones. The LHRH antagonist had no apparent effect on prolactin release.

It appears that the corpus luteum is relatively unresponsive to the high serum LH concentrations during the early luteal phase, but that responsiveness increases as the corpus luteum develops. The corpus luteum is, however, susceptible to withdrawal of LH not only in the mid–late luteal phase when the relationship with LH is apparent, but also during the early luteal phase.

J. Endocr. (1986) 111, 83–90

ABSTRACT

Chronic treatment with high doses of ACTH leads to marked reduction in aldosterone biosynthesis and secretion both in vivo and in vitro. In contrast, it has been reported that peripheral plasma aldosterone levels may be elevated following prolonged ACTH treatment. The present study attempts to determine the reason(s) for this apparently paradoxical finding. ACTH treatment (40 μg/100 g body weight) of male Sprague–Dawley rats for 7 days caused a decrease of more than 90% in aldosterone secretion into the adrenal vein in vivo and aldosterone production by intact adrenal capsules incubated in vitro. In contrast, peripheral plasma aldosterone levels appeared to be increased when measured by radioimmunoassay using two different polyclonal antibodies (antibody 1 (AB1) raised against aldosterone-3-carboxymethyloxime–bovine serum albumin (BSA) and antibody 2 (AB2) raised against aldosterone-21-hemisuccinate–BSA). However, when a highly specific monoclonal antibody (raised against aldosterone-3-carboxymethyloxime–BSA and showing low cross-reactivity to aldosterone metabolites) was used, peripheral plasma aldosterone levels appeared to be reduced in ACTH-treated rats. Following chromatographic fractionation of peripheral plasma, significantly more material with aldosterone-like immunoreactivity, but which was less polar than authentic aldosterone in chromatographic mobility, was detected in the fractions using antibodies AB1 and AB2. The absence of this material from fractions of adrenal vein plasma leads us to infer that this material is generated in the peripheral circulation, probably as a result of hepatic metabolism. In addition, the overall metabolic clearance rate (MCR) of [³H] aldosterone was found to be significantly decreased following prolonged ACTH treatment. We conclude that the seemingly discrepant findings with regard to the effects of chronic ACTH treatment on peripheral plasma aldosterone levels and the secretion of aldosterone in vivo can be reconciled by (1) the changes in the overall MCR of aldosterone and (2) the generation of increased quantities of aldosterone metabolites such as 5α-dihydroaldosterone and 3α,5β-tetrahydroaldosterone which show significant cross-reactivity with some aldosterone antibodies.

Journal of Endocrinology (1993) 137, 445–455

Bone morphogenetic proteins (BMPs) have been shown to influence the regulation of FSH synthesis and secretion at the level of the pituitary. Primary pituitary cells were harvested and cultured from Booroola ewes homozygous for a mutation in activin receptor-like kinase 6 (ALK6) also known as BMP receptor IB (BMPRIB), and from wild-type (WT) ewes to determine if the mutation caused alterations in FSH secretion in vitro. The cells were collected 24 h following induction of luteolysis and cultured for 72 h prior to being challenged for 24 h with BMP2, BMP4, BMP6, growth and differentiation factor-9 (GDF9), transforming growth factor-β 1, activin-A and GnRH. The levels of FSH and LH were measured by RIA and then compared with the untreated controls. Primary pituitary cell cultures from Booroola ewes secreted less FSH than WT cells in the presence of BMP2, BMP4 and BMP6. These BMPs did not affect the FSH stores within the cells, or the levels of LH released. GDF9 appeared to act in a BMP-like manner by suppressing FSH secretion. The ALK6 receptor however, was not found to co-localise with gonadotroph cells in either Booroola or WT pituitary tissues. These findings imply that the increased sensitivity of Booroola cells to BMP2, BMP4, BMP6 and GDF9 cannot be due to the direct action of the ALK6 mutant Booroola receptor in the cells that synthesise FSH.