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Substantial evidence shows that the hypophyseal–pituitary–adrenal (HPA) axis and corticosteroids are involved in the process of addiction to a variety of agents, and the adrenal cortex has a key role. In general, plasma concentrations of cortisol (or corticosterone in rats or mice) increase on drug withdrawal in a manner that suggests correlation with the behavioural and symptomatic sequelae both in man and in experimental animals. Corticosteroid levels fall back to normal values in resumption of drug intake. The possible interactions between brain corticotrophin releasing hormone (CRH) and proopiomelanocortin (POMC) products and the systemic HPA, and additionally with the local CRH–POMC system in the adrenal gland itself, are complex. Nevertheless, the evidence increasingly suggests that all may be interlinked and that CRH in the brain and brain POMC products interact with the blood-borne HPA directly or indirectly. Corticosteroids themselves are known to affect mood profoundly and may themselves be addictive. Additionally, there is a heightened susceptibility for addicted subjects to relapse in conditions that are associated with change in HPA activity, such as in stress, or at different times of the day. Recent studies give compelling evidence that a significant part of the array of addictive symptoms is directly attributable to the secretory activity of the adrenal cortex and the actions of corticosteroids. Additionally, sex differences in addiction may also be attributable to adrenocortical function: in humans, males may be protected through higher secretion of DHEA (and DHEAS), and in rats, females may be more susceptible because of higher corticosterone secretion.

Much evidence now suggests that angiotensin II has roles in normal functions of the breast that may be altered or attenuated in cancer. Both angiotensin type 1 (AT1) and type 2 (AT2) receptors are present particularly in the secretory epithelium. Additionally, all the elements of a tissue renin–angiotensin system, angiotensinogen, prorenin and angiotensin-converting enzyme (ACE), are also present and distributed in different cell types in a manner suggesting a close relationship with sites of angiotensin II activity. These findings are consistent with the concept that stromal elements and myoepithelium are instrumental in maintaining normal epithelial structure and function. In disease, this system becomes disrupted, particularly in invasive carcinoma. Both AT1 and AT2 receptors are present in tumours and may be up-regulated in some. Experimentally, angiotensin II, acting via the AT1 receptor, increases tumour cell proliferation and angiogenesis, both these are inhibited by blocking its production or function. Epidemiological evidence on the effect of expression levels of ACE or the distribution of ACE or AT1 receptor variants in many types of cancer gives indirect support to these concepts. It is possible that there is a case for the therapeutic use of high doses of ACE inhibitors and AT1 receptor blockers in breast cancer, as there may be for AT2 receptor agonists, though this awaits full investigation. Attention is drawn to the possibility of blocking specific AT1-mediated intracellular signalling pathways, for example by AT1-directed antibodies, which exploit the possibility that the extracellular N-terminus of the AT1 receptor may have previously unsuspected signalling roles.

Over the 70 or so years since their discovery, there has been continuous interest and activity in the field of corticosteroid functions. However, despite major advances in the characterisation of receptors and coregulators, in some ways we still lack clear insight into the mechanism of receptor activation, and, in particular, the relationship between steroid hormone structure and function remains obscure. Thus, why should deoxycorticosterone (DOC) reportedly be a weak mineralocorticoid, while the addition of an 11β-hydroxyl group produces glucocorticoid activity, yet further hydroxylation at C18 leads to the most potent mineralocorticoid, aldosterone? This review aims to show that the field has been confused by the misreading of the earlier literature and that DOC, far from being relatively inactive, in fact has a wide range of activities not shared by the other corticoids. In contrast to the accepted view, the presence of an 11β-hydroxyl group yields, in corticosterone or cortisol, hormones with more limited functions, and also more readily regulated, by 11β-hydroxysteroid dehydrogenase. This interpretation leads to a more systematic understanding of structure–function relationships in the corticosteroids and may assist more rational drug design.

Although the zonation of the adrenal cortex has a clear functional role, the mechanisms that maintain it remain largely conjectural. The concept that an outer proliferative layer gives rise to cells that migrate inwards, adopting sequentially the zona glomerulosa, fasciculata and reticularis phenotypes, has yet to be explained mechanistically. In other tissues, Eph receptor (EphR)/ephrin signalling provides a mechanism for cellular orientation and migration patterns. Real-time PCR and other methods were used to determine the possible role of Eph/ephrin systems in the rat adrenal. mRNA coding for several members of the EphR family was detected, but out of these, EphA2 provided the closest parallel to zonal organisation. In situ hybridisation showed that EphA2 mRNA and EphA protein were predominantly located in the zona glomerulosa. Its transcription closely reflected expected changes in the glomerulosa phenotype, thus it was increased after a low-sodium diet, but decreased by pretreatment with the angiotensin-converting enzyme inhibitor, captopril. It was also decreased by ACTH treatment, but unaffected by betamethasone. mRNA coding for ephrin A1, the major ligand for the EphA receptors, was also detected in the rat adrenal, though changes evoked by the various pretreatments did not clearly reflect the expected changes in zonal function. Because the maintenance of cellular zonation requires clear positional signals within the adrenal cortex, these data support a role for Eph forward and reverse signalling in the maintenance of adrenocortical zonation.

The extracellular N-terminus of G-protein-coupled receptors may be involved in signalling events. We examined this in the angiotensin II type 1 receptor (AT1-R) using monoclonal antibody 6313/G2, raised against a conserved sequence in the N-terminal domain, and found it evokes inhibitory and stimulatory responses. In rat aortic smooth muscle cell (RASMC) primary cultures, 6313/G2 (2.5 μg/ml) inhibited both basal and angiotensin II (Ang II; 10−7 mol/l)-stimulated [H3]thymidine incorporation. Exposure to 6313/G2 gave sustained increases in phosphorylated protein kinase Cα (PKCα) but gave a decrease in phosphorylated p44/42 extracellular signal-regulated kinases (ERK1/2) sustained from 10 min to 48 h compared with untreated control RASMC. In contrast, Ang II had no effect on PKCα, and, though it is acutely stimulatory (up to 5 min), it had no sustained effect on ERK1/2 either. Using Fura-2 and microfluorimetry, 6313/G2 added alone induced a transient increase in intracellular calcium ([Ca2+]i), with a characteristic response curve different from that of Ang II itself. The antibody was without effect on an Ang II-stimulated activator protein-1 reporter system, though it reduced unstimulated reporter activity. Such discriminatory effects on intracellular signalling suggest that the AT1-R N-terminus itself might be a target for therapeutic intervention in chronic vascular disease.

Normal pubertal development in humans involves two distinct processes: maturation of adrenal androgen secretion (adrenarche) and activation of the hypothalamic–pituitary–gonadal axis (gonadarche). One factor thought to contribute to the adrenarche in man is increased adrenal 17-hydroxylase (CYP17) activity. In the rat, there is evidence for adrenal involvement in the initiation of puberty, but the adrenal glands of this species are generally thought to express CYP17 only very poorly at best. To further examine the nature of postnatal adrenal development in rat, plasma samples and adrenal tissues were taken from animals aged 2–90 days, circulating adrenal steroids assayed, and adrenal zones assessed quantitatively. A relative increase in zona reticularis, and peaks of circulating cortisol, androstenedione, and 17-OH-progesterone were observed around postnatal days 16–20, clearly before the development of the gonads, which begins at 30–35 days. Quantitative reverse transcriptase PCR confirmed a peak in mRNA coding for CYP17 in adrenal tissue from rats of similar age. The results suggest that the rat adrenal has the capacity to secrete steroids arising from 17-hydroxylation, and that this may contribute to a process similar to human adrenarche.

The adrenal gland of the rat is continuously regenerated through proliferation of a stem cell population in the outer part of the gland. To clarify the location of proliferative events within the adrenal gland, and the factors that stimulate them, rat adrenal capsule preparations, consisting of capsule, zona glomerulosa (ZG) and the outer zona fasciculata (ZF) were maintained in vitro under different conditions of stimulation, for varying periods. Sites of proliferation were identified by 5-bromo-2′-deoxy-uridine (BrdU) staining, and the distribution of classical MAP kinase (MAPK) family members, extracellular signal-regulated kinase (ERK) 1 and 2, immunoreactivity was determined using immunocytochemistry. BrdU staining was limited to the outer glomerulosa and the capsule, where it was enhanced by angiotensin II, but not by a high potassium ion concentration nor by ACTH. In contrast, ERK1/2 immunoreactivity was distributed throughout the ZG and in the medulla, with none detectable in the ZF and reticularis. Furthermore, angiotensin II, potassium ions and ACTH were all shown to induce ERK1 and ERK2 phosphorylation in the ZG. Treatment of adrenal capsule tissue with the specific MAPK kinase inhibitor PD98059 revealed inhibition of ERK1/2 phosphorylation, but no effect on angiotensin II-induced aldosterone secretion. Although the distribution and activation of the MAPK pathway suggest a link with proliferation, the findings clearly designated only the outer part of the glomerulosa and capsule as a potential stem cell population. Further functions should be sought for the apparently silent major part of the glomerulosa.

Previous studies have shown the effects of angiotensin II (Ang II) in teleosts, and Ang II-binding sites have also been localized in tissues from rainbow trout. The purpose of this study was to extend these findings and to provide an analysis of Ang II receptor (Ang II-R) isoforms in three tissues obtained from European eel (Anguilla anguilla). Ang II-Rs were identified in eel liver, kidney and intestine membranes by the binding of either 0·5 nmol human 125I-labelled Tyr4-lle5-Ang II/l or increasing concentrations (1–120 nmol/l) of [3,5-3H]Tyr4-Ile5-Ang II. Using an isoelectric focusing technique, two Ang II-binding sites were identified in liver membranes. These migrated to isoelectric points (pI values) 6·5 and 6·7. Seventy per cent of binding to both sites was displaced by a 10 000-fold excess of unlabelled human Ang II. In both whole plasma membranes and brush border membranes from intestine, only one form of the Ang II-R was found, with pI 6·5 and high affinity (Kd=3·4 nmol/l) for the [3,5-3H]Tyr4-Ile5-Ang II. Similarly, only the isoform focusing at pI 6·5 was observed in renal tubular epithelial brush border membranes. Reduction of disulphide bridges with dithiothreitol significantly enhanced Ang II binding to the isoform at pI 6·5 in liver (PPP The data suggest the existence in eel liver of multiple forms of Ang II-R, which may have different functions, while one single form appeared to be present in enterocyte plasma membrane and in renal brush border membrane.

Adrenocortical regeneration after adrenal autotransplantation provides a model for the study of local autocrine/paracrine mechanisms involved in the growth and differentiation of the adrenal cortex. To study the possible involvement of some growth factors, namely basic fibroblast growth factor (bFGF, FGF-2) and insulin-like growth factor I (IGF-I), in cell differentiation, immunohistochemical and ultrastructural studies were carried out on adrenal autotransplants in adult male rats. To distinguish between fasciculata and glomerulosa-like cells with accuracy, tissue sections were immunostained with IZAb, which recognizes the inner zone antigen (IZAg) present in fasciculata and reticularis cells but absent from the glomerulosa, and by electron microscopy. IGF-I-treated animals exhibited a clear glomerulosa-like zone that was devoid of IZAb immunostaining. In this outer subcapsular area, ultrastructural examination showed cells containing mitochondria with irregular cristae resembling those of the fetal or immature glomerulosa cells. In contrast, no significant morphological differences were observed in bFGF-treated animals when compared with those from saline-treated controls, in both of which, IZAb immunostaining occurred in almost all adrenocortical cells, with no clear zonation or glomerulosa, as seen in the intact animal. Plasma aldosterone and corticosterone concentrations were lower in autotransplanted control animals than in intact controls, although plasma renin activities were similar. IGF-I treatment significantly increased aldosterone concentrations, whereas corticosterone and plasma renin activity were reduced. bFGF infusion further reduced plasma aldosterone, although plasma renin activity and corticosterone were unaffected. These results suggest that the two growth factors have different effects on zonal differentiation and function in the autotransplanted gland. In particular, bFGF, by reducing glomerulosa function, appears partly to replicate the actions of ACTH in normal animals. In contrast, IGF-I enhances the glomerulosa secreting phenotype and diminishes that of the fasciculata/reticularis, possibly replicating the actions of angiotensin II or a low sodium diet.

Evidence exists for the presence of a discrete tissue renin-angiotensin system (RAS) in mouse and rat pancreas that is thought largely to be associated with the vasculature. To investigate this in the human pancreas, and to establish whether the cellular sites of RAS components include the islets of Langerhans, we used immunocytochemistry to localise the expression of angiotensin II (AT1) receptors and (pro)renin, and non-isotopic in situ hybridisation to localise transcription of the (pro)renin gene. Identification of cell types in the islets of Langerhans was achieved using antibodies to glucagon and insulin. The results show the presence of the AT1 receptor and (pro)renin both in the beta cells of the islets of Langerhans, and in endothelial cells of the pancreatic vasculature. Transcription of (pro)renin mRNA, however, was confined to connective tissue surrounding the blood vessels and in reticular fibres within the islets. These findings are similar to those obtained in other tissues, and suggest that renin may be released from its sites of synthesis and taken up by possible cellular sites of action. The results presented here suggest that a tissue RAS may be present in human pancreas and that it may directly affect beta cell function as well as pancreatic blood flow.

The tissue renin-angiotensin systems (RAS) may have specific roles that complement those of the systemic RAS. In the adrenal, the tissue RAS has been implicated in the regulation of glomerulosa tissue growth and function, and in mediating the response of the tissue to stimulation by ACTH and potassium ions. To examine the role of the rat adrenal tissue RAS in its response to angiotensin II stimulation, adrenals were incubated either as bisected glands or as separated capsular glands (largely glomerulosa) under control conditions, or in the presence of the angiotensin-converting enzyme inhibitor captopril, or of angiotensin II, or both. Captopril inhibited the two different tissue preparations in different ways. In the capsular gland it inhibited basal aldosterone output, but facilitated its response to angiotensin II. In the bisected gland, captopril inhibited the response of aldosterone to angiotensin II. Other data suggest that one way in which captopril functions is by preventing the conversion of fasciculata-generated 18-hydroxydeoxycorticosterone (18-OH-DOC) to aldosterone in the glomerulosa. Immunolocalisation of 18-OH-DOC in perfused rat adrenal confirms that one function of angiotensin II is to mobilise tissue-sequestered 18-OH-DOC. The results illustrate the importance of tissue RAS in the synthesis of aldosterone and the response to angiotensin II.

Both [3H]noradrenaline ([3H]NA) and ATP were released in response to supramaximal electric field stimulation in superfused rat adrenal capsule-glomerulosa preparations. The voltage-dependent potassium channel blocker 4-aminopyridine enhanced, while the ATP-sensitive potassium channel blocker glibenclamide failed to affect the stimulation-evoked release of [3H]NA. The selective α2-adrenoceptor antagonist CH-38083 enhanced the evoked release of [3H]NA while the P2 receptor agonist ATP and α,β-methylene-ATP failed to affect it. Neither the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) nor the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) influenced the stimulation-evoked [3H]NA release. The data showed that ATP was released from capsule-glomerulosa preparations in response to field stimulation together with but independently from [3H]NA, and that the local noradrenergic varicose axon terminals are not equipped with purinoceptors sensitive to ATP and/or adenosine. High concentrations of ATP also stimulated steroid hormone secretion in vitro, and thus may have a physiological role in this tissue. The presence of ecto-Ca2+-ATPases, enzymes able to terminate the effect of ATP, was demonstrated around the nerve profiles at the border of the capsule and zona glomerulosa tissue. Journal of Endocrinology (1997) 153, 105–114

Immunocytochemistry of paraffin-embedded and cryostat sections of eel (Anguilla anguilla) gill showed that angiotensin II receptors (Ang II-R) were present in chloride cells, uniformly distributed in the cytoplasm and on surface membranes. Computerised image analysis of these preparations showed that gills from sea water (SW)-adapted animals had a significantly (3-fold) higher Ang II-R concentration compared with freshwater (FW)-adapted eel gills. Isoelectric focusing gel electrophoresis revealed two Ang II-R isoforms with pI 6·5 and 6·6 that were differentially modulated by environmental salinity: they were equally abundant in SW while in FW the pI 6·6/pI 6·5 ratio was 1·66. Using catalytic cytochemistry with image analysis, gill chloride cell membrane Na+/K+ATPase activity was shown to increase 4-fold in response to SW adaptation. Additionally, perfusion of gills for 30 min with 0·1, 10 or with 100 nM Ang II provoked a dose-dependent increment in Na+/K+ATPase activity in FW, and a biphasic response in SW gills in which activity was significantly increased at low Ang II concentrations but was reduced to basal values at 100 nM. The data suggest that adaptation to sea water significantly increases Ang II-R concentration in the chloride cell and, together with the effects of Ang II on Na+/K+ATPase activity, suggest a role for this hormone in gill NaCl retention. The different responses of Na+/K+ATPase to Ang II stimulation in FW and SW may be attributed to the presence of two receptor subtypes that are differently modulated by salinity and that have opposing effects on Na+/K+ATPase.

The pH-sensitive fluorescent dye, 2′,7′-bis-carboxy-ethyl-5,6-carboxyfluorescein acetoxymethyl ester, was used to examine the effects of fish or human angiotensin II (Ang II) on the activity of the basolateral located Na+/H+ antiporter in eel intestinal cell suspensions. Exposure of eel enterocytes to either hormone led to an increased activity of the antiporter. This time- and dose-dependent stimulatory effect was inhibited by the specific antiporter inhibitor dimethylamiloride (DMA). Preincubation with a monoclonal antibody (6313/G2), directed against the N-terminal extracellular domain of the mammalian AT1 Ang II receptor, prevented the stimulatory effect of the hormone and inhibited the binding of [3,5-3H]Tyr4-Ile5-Ang II to intestinal cell suspensions, suggesting specific binding of the antibody to the eel Ang II receptor. The results indicate that both fish and human Ang II stimulate the DMA-sensitive Na+/H+ antiporter present in eel intestinal cells by means of a mammalian AT1-like receptor.

We evaluated the presence and variability of oestrogen receptor (ER) isoforms in endometrial cancer by using [3H]oestradiol-labelled ERs and the H222 monoclonal antibody obtained from the Abbott enzyme immunoassay kit. Using isoelectric focusing (IEF), endometrial ER was shown to be composed of four different species, with pI values of 6·1, 6·3, 6·6 and 6·8, indistinguishable from the isoforms found in normal rat uterus, and human breast and larynx carcinomas. The isoforms at pI 6·3, 6·6 and 6·8, all sedimenting at 4S by sucrose gradient fractionation, showed, on two-dimensional SDS electrophoresis, relative masses of 50, 70 and 65 kDa respectively, equal to the masses previously found in breast cancer. These isoforms did not alter their pI values during IEF fractionation performed in a linear gradient of urea, while the pI 6·1, sedimenting at 8S, generated a new isoform at about 9 mol/l urea with pI 7·2 and a relative mass of 65 kDa. The urea-dissociated isoform (pI 7·2) was able approximately to double the antibody binding with respect to the non-dissociated oligomer, which suggested that some epitopes are 'masked', i.e. not accessible to the antibodies when ER is present in its complexed form. The evidence thus suggested that the oligomer at pI 6·1 contained a single 65 kDa ER form which, as a monomer, focused at pI 7·2. The variability in the ER isoform profile found in endometrial cancer was similar to the variability previously reported in breast and larynx carcinomas. The balance between these isoforms could be a dynamic parameter involved in the functionality of this receptor and consequently in cell transformation.

This study located the particular cell types involved in the synthesis of growth factors in adult female rat adrenal glands. Non-isotopic in situ hybridization was used and the cellular localizations of the mRNAs of basic fibroblast growth factor (bFGF), IGF-I, and transforming growth factor-β1 (TGF-β1) were studied in adrenals from control animals and from those treated with ACTH or subjected to dietary sodium restriction. The adrenal medulla was the richest source of both bFGF and IGF-1 mRNA in both control and experimental rat adrenals. In the cortex, bFGF and IGF-I mRNAs were found mainly in the zona fasciculata in control animals, although some transcription was also detected in the zona reticularis and zona glomerulosa. Both ACTH and sodium restriction activated bFGF and IGF-I gene expression in the zona glomerulosa. Since cellular proliferation and differentiation occur primarily in the outer cortex, the data are consistent with the view that bFGF and IGF-I act as an autocrine/paracrine mitogen and differentiation regulator respectively in the rat adrenal cortex. Very small amounts of TGF-β1 mRNA were detected, predominantly in the zona fasciculata of control rats. There were no observable differences in amounts and localization of TGF-β1 mRNA between the adrenals of control rats and those treated with ACTH for 1 day. TGF-β1 mRNA was very weak or undetectable in the adrenals from rats treated with ACTH for three and five days or from sodium-restricted rats. Although TGF-β1 immunoreactive protein has been shown to be present in the zonae fasciculata and reticularis and to modulate negatively the steroidogenic activities in the adrenal cortex of other species, its gene is not actively expressed in rat adrenals. The present results showed that ACTH administration or dietary sodium restriction, both important adrenal mitogens in vivo, significantly altered the spatial patterns of the distribution of bFGF and IGF-I mRNAs and also increased the amount of bFGF mRNA in the adrenal cortex. This suggests that growth and differentiation of the adrenal cortex are partly mediated by bFGF and IGF-I. Journal of Endocrinology (1995) 144, 379–387

Several lines of experimentation suggest that a tissue-sequestered pool of 18-hydroxydeoxycorticosterone (18-OH-DOC) in the rat adrenal may be mobilized as an aldosterone precursor. We show here that this steroid is maintained in a non-extractable form in the membranes of collagenase-dispersed fasciculata/reticularis cells. Because of this stability, the complex can be identified by immunocytochemistry and also, in IEF gels of solubilized inner adrenocortical zone membrane preparations, by immunoblotting. However, the complexed steroid cannot be extracted from the gels into organic solvent unless first treated with trypsin. Preincubation of viable whole glandular tissue with trypsin significantly enhanced aldosterone output and eliminated the trypsin-releasable 18-OH-DOC pool in IEF gels of solubilized inner zone membranes. Both prior sodium depletion and acute trypsin stimulation of whole glands enhanced extractable 18-OH-DOC in glomerulosa tissue membranes. Other experiments using in situ hybridization show that mRNA coding for 11β-hydroxylase (which generates 18-OH-DOC) is confined to the inner adrenocortical zones, whereas aldosterone synthase (which does not) is transcribed exclusively in the glomerulosa. The data suggest that a pool of 18-OH-DOC in inner zone membranes can be mobilized for utilization as an aldosterone precursor in the glomerulosa. The results also indicate the existence of an entirely novel tightly binding steroid carrier from which steroid cannot be extracted by organic solvent unless first subjected to proteolytic degradation. Journal of Endocrinology (1995) 144, 359–368

Isoelectric focusing analysis showed a single angiotensin II (All)-receptor complex migrating to pI 6·8 in nuclear preparations, while in plasma membranes a charge heterogeneity of the All receptor subtype AT1 was observed. 125I-Labelled All binding sites were found in intact nuclei and were not detected in nuclear extracts. Neither disruption of cytoskeletal elements by colchicine nor prevention of endosome acidification by chloroquine had any effect on nuclear accumulation of AIL Nevertheless, the monovalent ionophore monensin inhibited nuclear accumulation of 125I-Labelled All. Our findings are consistent with the hypothesis that processing through the Golgi apparatus could be involved in the nuclear accumulation of AIL Journal of Endocrinology (1994) 143, 449–453

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

The intracellular mechanisms of action of alpha-MSH in rat adrenocortical cells were examined. When rat adrenal capsule (largely glomerulosa) cells were stimulated with a range of concentrations of alpha-MSH there was significant stimulation of aldosterone secretion at 10(-10) mol/l, although cyclic AMP was not increased until high concentrations of alpha-MSH were used (10(-6) mol/l and above). However, cells incubated with ACTH showed an increase in aldosterone secretion at 10(-11) mol/l and levels of cyclic AMP were elevated at 10(-9) mol ACTH/l. When rat adrenal whole capsules were incubated with alpha-MSH, membrane-bound protein kinase C (PKC) activity was increased and cytosolic enzyme activity decreased, showing PKC activation. Stimulation with angiotensin II also induced translocation of PKC activity, but ACTH did not. When [3H]inositol-loaded glomerulosa cells were stimulated with alpha-MSH there was significant generation of [3H]inositol trisphosphate (IP3) at concentrations of alpha-MSH which stimulated secretion of aldosterone. Significantly increased levels of [3H]IP3 were also measured when loaded cells were exposed to angiotensin II. ACTH did not cause any significant stimulation of [3H]IP3 production at any concentration used. These results indicate that activation of PKC and phospholipase C is important in modulating the steroidogenic effect of alpha-MSH.

The rat adrenal cortex contains quantities of dopamine that are compatible with its function as a neurotransmitter, suggesting that locally released dopamine may act as a neuroregulator within the gland. This possibility has been tested by comparing the effects of dopamine on aldosterone secretion in the perfused adrenal with the effects of stimuli designed to provoke the release of intraglandular dopamine. Infusion of dopamine (0·1–100 μmol/l for 10-min periods) into the isolated perfused rat adrenal gland resulted in a transient, dose-related reduction of aldosterone secretion to a minimum of approximately 50% of the basal value at 1 μmol dopamine/l (ratio of experimental to control measurements, R = 0·53 ± 0·06 (s.e.m.); n = 5). In contrast, dopamine (1–100 μmol/l) had no effect on aldosterone production by dispersed zona glomerulosa cell preparations incubated in vitro. The effects of changes in K+ concentration (3·9–52 mmol/l) on aldosterone secretion in the perfused gland and dispersed cell preparations were also compared. A similar bell-shaped dose–response relationship was seen in both preparations between 6 and 32 mmol K+/l, with a maximum at 8·4 mmol K+/l and a return to control values with 16, 24 or 32 mmol K+/l. However, infusion of media with very high K+ concentrations (42 or 52 mmol K+/l) reduced the secretion of aldosterone by the perfused gland to approximately 50% of the basal value (R = 0·51 ± 0·05, n = 9; R = 0·49± 0·08, n = 9; respectively) but produced no change in aldosterone production by zona glomerulosa cells. Electrical field stimulation (pulse width 1 ms, 1 Hz at 60 V for 5 min) of the perfused gland also resulted in a reduction in aldosterone secretion (R = 0·66 ± 0·66, n = 6). In the presence of 1 μmol haloperidol/l, a dopamine antagonist, no effect on aldosterone secretion was seen under control conditions, but the responses to 1 μmol dopamine/l, 52 mmol K+/l and field stimulation were eliminated. The results are consistent with the view that aldosterone secretion by the perfused adrenal gland is subject to an inhibitory dopaminergic control, which may originate from catecholaminergic neurones within the gland itself. Journal of Endocrinology (1992) 133, 275–282

It is now known that in the rat there are two distinct species of cytochrome P45011β/18, namely aldosterone synthase and 11β-hydroxylase. Whereas aldosterone synthase is located exclusively in the zona glomerulosa, the zonal distribution and site of production of 11 β-hydroxylase is not entirely clear. In the present study we examined the zonal expression of 11β-hydroxylase mRNA in adrenals from control rats and animals subjected to ACTH treatment and dietary sodium restriction using a non-isotopic in-situ hybridization technique. The results were compared with those obtained using an inner zone specific antigenic (IZAg) marker to give unequivocal identification of the adrenocortical cell types. 11 β-Hydroxylase mRNA was clearly shown to be expressed in the inner zones of the control rat adrenal cortex, and none was detected in the zona glomerulosa and medulla. The message was more abundant in the outer zona fasciculata. A similar pattern of distribution of 11β-hydroxylase mRNA was observed in adrenals from rats subjected to dietary sodium restriction, although the width of the negatively staining layer of zona glomerulosa was significantly increased. In rats treated with 100 μg ACTH for 1 day, the number of cells expressing 11β-hydroxylase mRNA was increased, especially in the zona reticularis. With continued ACTH treatment, 11β-hydroxylase mRNA was found in the region of the zona glomerulosa, and after 3 and 5 days of ACTH treatment cells expressing 11β-hydroxylase mRNA extended to the connective tissue capsule. At this time there was a significant reduction in the total expression of the message compared with the controls. These results suggest that the presence of 11β-hydroxylase in the zona glomerulosa cells is not essential for the late pathway for aldosterone biosynthesis from deoxycorticosterone. Like IZAg, the distribution of 11β-hydroxylase mRNA after prolonged ACTH treatment provides further evidence to support the hypothesis that ACTH increases the conversion of zona glomerulosa to zona fasciculata-like cells. Journal of Endocrinology (1993) 139, 301–306

The effect of supramaximal electric field stimulation on [3H]noradrenaline (NA) release and hormone production by rat adrenal capsule-glomerulosa preparations was studied using a microvolume perfusion system. A substantial proportion (about 20%) of nerve endings (varicosities) were observed close to zona glomerulosa cells, and about half of them appeared to be catecholaminergic, as judged by the chromaffin reaction of the synaptic vesicles studied at electron microscopic level. In tissue, preloaded with [3H]NA, the release of NA in response to electrical stimulation was frequency-dependent. Reserpinization, calcium removal or inhibition of Na+ influx by tetrodotoxin completely blocked NA release by field stimulation, indicating that the release resulted from axonal activity and is of vesicular origin. Neither the α2-adrenoceptor agonist xylazine nor the muscarine-receptor agonist oxotremorine affected the stimulation-evoked release of [3H]NA, suggesting that, in contrast with other neurones present in the central nervous system or in the peripheral autonomic nervous system but like those in the median eminence, these axon terminals contained few presynaptic modulatory receptors. The NA (10·20 ± 1·79 (s.e.m.) μg/g, n = 9), adrenaline (24·38 ± 5·50 μg/g, n = 9) and dopamine (0·35 ± 0·09 μg/g, n = 6) contents of the preparations were high, as determined by high-performance liquid chromatography. Our observations that the release and content of NA is high, and that a substantial proportion of catecholaminergic axon terminals lie in close proximity to zona glomerulosa cells (median value of the distance 300 nm) or to smooth muscle cells of the vessels, suggest that NA released from local adrenergic neurones without being presynaptically modulated may play an important role in finetuning both steroid production and/or blood flow through the gland, itself a powerful modulator of the adrenocortical response. This local modulating effect of NA may be especially significant when sympathetic activity is enhanced. Journal of Endocrinology (1992) 135, 551–561

The finding that incubation of rat adrenal capsules (largely zona glomerulosa) with trypsin reproducibly releases aldosterone and 18-hydroxycorticosterone (18-OH-B) from tightly protein-bound tissue stores leads to the hypothesis that the secretion of these steroids may be under the control of endogenous proteases. Rat adrenal capsule whole tissue and collagenase-dispersed cells were incubated under conditions of stimulation by (1–24)ACTH (10−7 mol/l), potassium (8·4 × 10−3 mol/l) or dibutyryl cyclic AMP (dbcAMP) (10−4 mol/l) with the addition in some flasks of one of the following protease inhibitors at the appropriate concentration for their known actions: Nα-p-tosyl-l-arginine methyl ester (TAME; 10−2 mol/l), 2-nitro-4-carboxyphenyl-N,N′-diphenylcarbamate (NCDC; 2×10−6 mol/l), Nα-benzoyl-l-arginine (BA; 10−2 mol/l), p-nitrophenyl-Nα-benzyloxycarbonyl-l-lysinate (CBZ-NL; 2×10−6 mol/l) and soybean trypsin inhibitor (STI; 1 mg/ml). The (1–24)ACTH-stimulated steroid output in dispersed cells was not affected by NCDC, BA or CBZ-NL. However, all of the inhibitors except STI produced selective effects on aldosterone and 18-OH-B production by whole capsule tissue under certain conditions. Thus TAME and NCDC significantly inhibited the dbcAMP-stimulated production of these two steroids (aldosterone values decreased from 328±35 to 128±15 and 157±32 ng/gland pair respectively) and furthermore NCDC elicited the same effect in potassium- or ACTH-stimulated whole tissue (e.g. in K+-stimulated tissue aldosterone decreased from 79±15 to 40±7 ng/gland pair). The reverse effect was shown by BA and CBZ-NL in potassium-stimulated whole tissue, and yields of aldosterone and 18-OH-B were significantly enhanced (aldosterone increased from 79±15 ng/pair to 151±14 ng in the presence of BA). The high molecular weight inhibitor STI had no effect on potassium-stimulated whole tissue, but enhanced the yield of extractable aldosterone from 9·±1·7 to 16·9±2·3 ng/pair when added to incubations of a cytosol preparation. The results suggest that endogenous proteases in rat adrenal whole capsule tissue play specific roles in the control of aldosterone and 18-OH-B secretion. Some (type 1) whose action is mimicked by trypsin, are inhibited by TAME and NCDC and appear to be involved in the release of these two steroids from their tight (apparently covalent) binding to protein. Others (type 2) are inhibited by BA, CBZ-NL and STI, and one interpretation of their function is that they are concerned with the activation of type 1 proteases. The mechanisms envisaged provide a selective means for control of secretion of the late-pathway steroid products in the rat adrenal zona glomerulosa. These mechanisms are not apparent in dispersed cell incubations, probably because of the loss of steroid–protein complexes in these preparations.

A technique for the perfusion of the rat adrenal cortex is described. With tissue culture Medium 199 the preparation was responsive in terms of steroid production to both ACTH and K+ ions. Production of corticosterone and 18-hydroxydeoxycorticosterone (18-hydroxy-DOC) was stimulated by ACTH when it was administered at rates between 5 μu./min and 5 mu./min. Increasing the K+ ion concentration of the perfusate from 3·6 to 5·4 and 8·9 mmol/l stimulated the production of aldosterone, 18-hydroxycorticosterone and deoxycorticosterone, although not of corticosterone or 18-hydroxy-DOC. This preparation has been used to study further the mechanism of secretion of corticosterone and 18-hydroxy-DOC. Thus, production of these two steroids was measured at different perfusion flows, varying between 0·1 and 0·6 ml/min, with different levels of ACTH stimulation. Corticosterone production was significantly (P < 0·001) increased by increasing flows both under control conditions and when ACTH was administered at constant rates of 50 μu./min or 1 mu./min. Production of 18-hydroxy-DOC was not affected by flow either under control conditions or with 50 μu. ACTH/min. However, when ACTH was administered at 1 mu./min, 18-hydroxy-DOC production was also significantly (P < 0·001) increased by flow. The results are consistent with those obtained in previous in-vitro studies and have been interpreted as suggesting that the main mechanism of corticosterone secretion is simple diffusion. In contrast, 18-hydroxy-DOC secretion, at least at sub-maximal levels of stimulation, appears to require a more complex process.

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

SUMMARY A kinetic study of corticosterone recovered during incubation of rat adrenal tissue with [4-14C]progesterone and [16-3H]pregnenolone shows that both its 3H: 14C ratio and its specific activity change with time throughout incubation. Corticosterone is itself metabolized, and hypotheses based on the rate of production of corticosterone may be therefore deceptive when the amount is measured at a single point in time. Thus disparities between the specific activities of products and intermediates may be expected which alone do not necessarily indicate differences between the biosynthetic pathways involved in production from endogenous precursors and those from added radioactive precursors. Other experiments suggest that steroids in incubation media, in concentrations comparable with those found in adrenal venous plasma, do not inhibit the continued synthesis of hormones.

SUMMARY Using incubation techniques, with progesterone as precursor, the capacity of the mouse ovary to form steroids was examined. Ovaries were taken from three groups of mice: (1) immature mice, (2) mature virgin females, (3) primigravid animals. The formation of the following steroids was suggested by the analytical methods used: 16-oxooestrone, 17-epioestriol, 17α-hydroxyprogesterone, 20α-hydroxyprogesterone, androstenedione, testosterone. Yields were very small from the ovaries of immature mice. The yields of oestrogens were greatest from ovaries of mature virgin females and of testosterone from those of pregnant mice. The factors contributing to the disappearance of the X zone of the adrenal gland during first pregnancy are discussed. It is concluded that one major factor is the secretion of testosterone by the ovary of the gravid mouse.

Mast cells were identified in the rat adrenal gland, located in the walls of arterioles at the point at which they penetrate the connective tissue capsule. The mast cell products, histamine and serotonin, both caused dose-dependent increases in rates of perfusion medium flow and steroid secretion in the isolated, perfused rat adrenal gland in situ. Compound 48–80, a mast cell degranulator, caused a significant increase in perfusion medium flow rate and steroid secretion by the in-situ perfused rat adrenal. Administration of disodium cromoglycate, a mast cell stabilizer, before administration of ACTH(1–24) virtually abolished the normal flow rate increment and significantly attenuated the corticosterone secretory response to ACTH(1–24). These observations strongly suggest that adrenal mast cells modulate both vascular and secretory responses in the intact adrenal gland of the rat. Journal of Endocrinology (1989) 121, 253–260