G. P. Vinson

Oxytocin and arginine vasopressin stimulate steroid secretion by the isolated perfused rat adrenal gland

Using the intact isolated perfused rat adrenal preparation we have shown for the first time a direct effect of oxytocin on adrenocortical steroid secretion. Oxytocin specifically stimulated aldosterone secretion in a dose-dependent manner with a threshold dose of 1 pmol. Arginine vasopressin was also shown to be a potent stimulus to aldosterone secretion and was additionally found to stimulate inner zone function. Using superfused adrenal cells, the effects of arginine vasopressin were only seen at 10,000 times higher doses than were effective in the intact perfused gland, and oxytocin had no effect at any dose. These results reinforce the hypothesis that tissue integrity is essential for full expression of steroidogenic control mechanisms. We conclude that oxytocin and vasopressin may play a role in the control of steroidogenesis.

Calcitonin gene-related peptide stimulates adrenocortical function in the isolated perfused rat adrenal gland in situ

Using the isolated perfused in situ rat adrenal preparation, we have shown a direct stimulatory action of calcitonin gene-related peptide on aldosterone secretion. The threshold dose for this action was 1 pmol, given as a bolus in 200 ul. CGRP also caused vasodilation in the adrenal gland, reflected in increased perfusate flow, and also stimulated corticosterone secretion, with a threshold of 0.1 pmol. Addition of CGRP to incubations of collagenase-dispersed adrenocortical cells had no effect on steroidogenesis. These results support the contention that CGRP, which have been identified in nerve terminals in the zona glomerulosa of the rat adrenal cortex, may have a role in the control of steroid secretion.

Corticosteroid biosynthesis from pregnenolone and progesterone by human adrenal tissue in vitro. A kinetic study.

Abstract Using a kinetic approach, a study has been made of the pathways of corticosteroid biosynthesis from 3 H -pregnenolone and 14 C — progesterone in human adrenal tissue. In the adult, the dissimilarity between the 3 H — and 14 C -product yield-time curves suggested that the two precursors were handled quite differently. In particular it appeared that 17-hydroxypregnenolone, which bore only the 3 H label, acted as an intermediate in the formation of 3 H -cortisol from 3 H -pregnenolone whereas only slight amounts of 3 H -progesterone were formed. From 14 C -progesterone, the formation of 14 C -corticosterone and 14 C -cortisol proceeded via the ‘classical’ pathways. In the foetus, the lack of 17-hydroxypregnenolone dehydrogenase seems to account for the relatively minute yields of 3 H -corticosteroids from 3 H -pregnenolone. The pattern of 14 C -progesterone metabolism in the foetus was the same as in the adult. These results are in agreement with the general conclusions reached by other authors in non-kinetic studies with human adrenal tissue, but are in striking contrast with the results obtained with adrenal tissue from other species.

Corticosteroid production in vitro by adrenal tissue from rats with inherited hypothalamic diabetes insipidus (brattleboro strain)

Abstract Steroid profiles formed by adrenal tissue from rats of the Brattleboro strain homozygous for the recessive gene causing diabetes insipidus (DI rats) were compared with those formed by glands from heterozygotes (non-diabetic: non-DI) during incubation in vitro. The most striking observation was that glands from DI rats showed a greatly reduced capacity to produce certain steroids with mineralocorticoid activity, particularly deoxycorticosterone (DOC) and 18-hydroxy-DOC (18-OH-DOC), and to a lesser degree, aldosterone (ald). Corticosterone (B) and 18-hydroxy-B were produced in similar amounts by glands from DI and non-DI animals. The impairment of DOC and 18-OH-DOC production in adrenals from DI animals was a feature of both capsule (mostly zona glomerulosa) and inner zone incubations. In incubations of tissue from DI animals, the addition of ACTH, or a low concentration of angiotensin amide to the incubation media stimulated corticosterone alone in inner zone incubations, but was without effect on other steroids, or on capsule incubations: with non-DI tissue, ACTH (but not the low concentration of angiotensin) stimulated corticosterone production, but again there was no effect on other steroids, or on the glomerulosa. Higher concentrations of angiotensin, or the addition of LH had only marginally significant effects. Addition of a “physiological” concentration of ADH to adrenal tissue from normal Wistar rats had no effect on the steroid profile. The results suggest the existence of as yet unidentified adrenocortical stimulators or inhibitors which exert effects on production of specific steroids, especially DOC and 18-OH-DOC, and which are effective both on the zona glomerulosa and on the inner adrenocortical zones.

Steroid profiles formed by rat adrenocortical whole tissue and cell suspensions under different conditions of stimulation

Abstract The production of five steroids, corticosterone, deoxycorticosterone (DOC), 18-hydroxydeoxycorticosterone (18-OH-DOC), 18-hydroxycorticosterone (18-OH-B) and aldosterone, has been measured following incubations of rat adrenal tissue. The following tissue preparations were used: whole gland minces, whole glands quartered, capsules and inner zones intact, or as cell suspensions. Stimulation by ACTH, LH and potassium was also studied. The steroid profile elaborated by the tissue varies in a striking manner depending both on the method of tissue preparation and on the mode of stimulation. In inner zones the variation is found in the relative amounts of corticosterone and 18-OH-DOC produced. In unstimulated cell suspensions, or in whole tissue subjected to preincubation, the corticosterone/18-OH-DOC ratio is less than 1, possibly as little as 0.2. After stimulation, or in other preparations the ratio can rise to as much as 1.6. In capsules, the most important effect is on the capacity to produce the 18-oxygenated steroids as a whole. Even under conditions of maximal stimulation by ACTH or potassium, this capacity is greatly impaired in cell suspensions compared with whole tissue, while corticosterone is unaffected. The findings are not compatible with the theory that ACTH (or other stimulants) act at a single site in the biosynthetic pathway. They are compatible with the view put forward from these laboratories over the years, that control of steroidogenesis is also effected through maintenance of separated pools of steroid within the tissue which have different metabolic fates. In particular it appears that the 18-oxygenated steroids (in contrast to corticosterone) may be sequestered within tissue stores, and that in part their secretion is controlled by release from such stores.

α-MSH and zona glomerulosa function in the rat

The responses of rat adrenal zona glomerulosa cells to stimulation by alpha-MSH and ACTH and related peptides have been studied. The major findings were that: (1) alpha-MSH stimulated corticosterone production in glomerulosa cells from from normal animals at concentrations of about 10(-10) mol/l, but other steroids, including aldosterone, were not significantly stimulated until levels of 10(-7) mol/l were used. Peptide structure-function relationships showed that in the adrenal cortex, in contrast with other systems, ACTH 4-10 had no effect and did not block the response of glomerulosa cells to alpha-MSH, bisacetyl Ser 1-alpha-MSH, (nor-valine-12)-alpha-MSH, and ACTH 1-13 amide were equipotent with alpha-MSH, while alpha-MSH 1-10 had activity but was considerably less potent. alpha-MSH 6-13, 7-13, 8-13 and lys-11-acetyl-alpha-MSH were all inactive. N-formyl-N-epsilon-benzyloxycarbonyl alpha-MSH stimulated only at 10(-6) mol/l. (2) Normalised alpha-MSH dose-response curves for aldosterone production in glomerulosa cells from normal rats, and corticosterone in inner zone cells were coincident. In glomerulosa cells, prior sodium depletion shifts the dose-response curve for aldosterone to the left, indicating a more sensitive response, and for corticosterone to the right. Bromocriptine treatment (which depresses the level of alpha-MSH in circulating plasma) and metoclopramide (which enhances it) respectively increased and decreased the sensitivity of the response of corticosterone to alpha-MSH in subsequently incubated glomerulosa cells, but had no effect on aldosterone. (3) In contrast, normalised ACTH stimulated dose-response curves for glomerulosa corticosterone and aldosterone, and for fasciculata corticosterone production were all coincident, and were unaffected by sodium depletion, or by metoclopramide or bromocriptine pretreatment. (4) Cyclic-AMP production by glomerulosa cells was stimulated by alpha-MSH only at levels of in excess of 10(-5) mol/l, five orders of magnitude greater than required to produce significant corticosterone stimulation. Under cyclic-AMP stimulation, the normalised responses of glomerulosa corticosterone and aldosterone, and of inner zone corticosterone were all coincident. The data suggest that alpha-MSH at low concentrations (less than 10(-7) mol/l) interacts with a glomerulosa cell receptor which is distinct from the ACTH receptor but interacts with the ACTH receptor at concentrations greater than 10(-5) mol/l. Corticosterone production is stimulated by alpha-MSH in cells from normal animals at concentrations within the normal range for circulating plasma (approximately 3 X 10(-10) mol/l), while aldosterone is stimulated by similar concentrations of alpha-MSH in cells from sodium depleted animals. The effects of sodium depletion are not modulated through changes in plasma alpha-MSH levels. At low concentrations alpha-MSH stimulation of glomerulosa cells is unlikely to be modulated by cyclic-AMP as second messenger.

Steroid 17-hydroxylation and androgen production by incubated rat adrenal tissue.

Rat adrenal tissue was decapsulated, and capsules (considered to consist largely of zona glomerulosa) and inner zones were separately incubated with [4-14C]-progesterone. Testosterone was found to be formed by both tissue types in yields at least 1% of those of corticosterone, confirming earlier observations. In addition, evidence was found for the production of radioactive 17-hydroxyprogesterone. androstenedione, 11-deoxycortisol and cortisol, in addition to the usual 17-deoxysteroids. Yields of cortisol were up to 20% of those of corticosterone, yields of the other steroids were about the same as testosterone. No significant differences in the yields of these products were found in capsule and inner zone incubations. From endogenous precursors, cortisol was again found to be formed in similar amounts by both tissue types. In some incubations, testosterone was apparently produced in larger amounts by the capsules, in others amounts were similar in capsules and inner zones. Addition of ACTH (20 mU per ml) increased the yields of testosterone in capsules but not inner zones. Sodium depletion had no effect on testosterone production. It is concluded that 17-hydroxylation can and does occur in the rat adrenal cortex, and it is likely that the pathways for the formation of cortisol and the androgens are similar to those described for other tissues. It is unlikely that 17-hydroxylation occurs in the cells of the glomerulosa, but the evidence suggests that it may be prominent in a region of the fasciculata directly underlying the glomerulosa, in the position of the zona intermedia.

Studies on the mechanism of secretion of rat adrenal steroids in vitro.

Abstract The possibility that in the rat adrenal cortex synthesis and secretion of steroid hormones may be distinct. processes has been studied using in vitro methods. The uptake and subsequent release of radioactive corticosterone and 18-OH-DOC added to incubation media showed little difference between the two steroids. Both were taken up rapidly by the tissue, and after exchange of medium, 50% was released within 15 min. The properties of muscle and adrenal tissue were qualitatively similar in these respects, and were consistent with steroid movement by diffusion. Different results were obtained when steroids formed from the tissues endogenous precursor supply were studied. Following incubation for 2h tissue and medium steroid cohtent were measured, and tissue/medium ratios of steroid were used as an index of secretion. With varying volumes of incubation medium (1.4–10 ml), secretion of corticosterone was related to volume, although synthesis was constant. With 18-hydroxydeoxycorticosterone (18-OH-DOC) on the other hand neither synthesis nor secretion was affected by medium volume. The effects of ouabain (10 −4 M) on synthesis and secretion were also studied, using a 5 ml volume of medium. Under these conditions, tissue levels of corticosterone were very low, while 18-OH-DOC was extractable from tissues in amounts up to 30% of the total synthesised. In capsules, steroid synthesis was decreased by ouabain, or by a lower potassium medium (3.6 mM) or both together. Secretion of corticosterone was not affected by these treatements, whereas secretion of 18-OH-DOC and aldosterone was greatly inhibited: maximally 50% of the synthesised 18-oxygenated steroid was retained within the tissue. These treatments also affected inner zone function, ouabain inhibited steroid synthesis, but the low potassium medium did not. Secretion of corticosterone was unaffected, whereas secretion of 18-OH-DOC was again inhibited both by low potassium medium and by ouabain. Maximally as in the capsules, 50% of the synthesised 18-OH-DOC was retained within the tissue. The results suggest that the secretion of 18-OH-DOC formed from endogenous precursors by rat adrenal tissue is controlled by a mechanism involving a ouabain sensitive process in the cell membrane. Corticosterone is released by a different mechanism, quite possibly passive diffusion.

Integrinβ1-mediated invasion of human breast cancer cells: anex vivo assay for invasiveness

BackgroundThe integrin cell adhesion molecule (CAM) family is intimately involved in cell adhesion and invasion through tissue basement membranes (BM). As a consequence of the short survival of patient-derived human breast cancer cells, the invasion of such cells has not been previously reported. Our aims were to optimise culture conditions in order to establish a reliable invasion assay and to assess the effect on invasion of perturbations of theβ1 integrin receptors.MethodsPure suspensions of viable carcinoma cells were isolated immunomagnetically from human breast cancer (HBC) samples and introduced onto a replicated glycoprotein BM within an invasion chamber. Degree of invasion was compared to bothβ1 integrin expression and tumour grade. Additionally, the effect ofβ1 receptor blockade with monoclonal antibody (mAb) was assessed.ResultsInvasion was significantly greater in grade II than grade III tumour cells (P=0.0012). Antiintegrinβ1 monoclonal antibody inhibited cancer cell invasion by a mean of 83.96 ± 4.80%.ConclusionsThe invasion assay confirmed the fundamental importance ofβ1 integrin receptors to transmembrane invasion and reports this for the first time in cells isolated from primary breast cancer. It represents a potent research tool for investigation of the tumour biology of invasion at the integrinβ1-mediated cell-basement membrane interface. This assay has the potential clinical application of improved stratification of patients for adjuvant therapy on a more individual basis than currently available.

Discrepancies between antibody (EIA) and saturation analysis of oestrogen receptor content in breast tumour samples

The use of different techniques for assay of oestrogen receptors (ER) in breast cancer raises the question of their relative effectiveness in measuring concentrations of functional receptors. Data were obtained on soluble receptors from supernatants from 58 primary breast tumour homogenates, using the ligand ([3H]oestradiol) binding assay with dextran-coated charcoal (DCC) separation, either at a single saturating ligand dose, or by Scatchard analysis, and by using the Abbott enzyme immunoassay (EIA) kit. As previous reports have shown, the two methods gave reasonably good correlation (r = 0.8), but EIA values were systematically higher than DCC (slope = 3.0). Similar values were obtained when the ER + ve/progesterone receptor (PR) + ve subgroup were examined separately (n = 34, r = 0.86, slope = 3.0). However the two sets of data were in much better agreement in the ER + ve/PR - ve subgroup (n = 10, r = 0.98, slope = 1.24). When analysed by isoelectric focusing on polyacrylamide gels (IEF), two major specific binding components were identified, at pI 6.1 and at pI 6.6. Both isoforms were present in 50/66 ER + ve PR + ve breast tumour samples, but only the pI 6.6 (4S) was present in most ER + ve/PR - ve samples (13/20). It appears that, compared with DCC, the EIA method gives much higher values for the 8S isoform, whereas the two methods detect the 4S isoform with similar sensitivity. In assays on the tumour cell lines, T47D and MCF-7, still greater discrepancies, at least 10-fold, were found between EIA and DCC data.