Search

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.

The standard model of adrenocortical zonation holds that the three main zones, glomerulosa, fasciculata, and reticularis each have a distinct function, producing mineralocorticoids (in fact just aldosterone), glucocorticoids, and androgens respectively. Moreover, each zone has its specific mechanism of regulation, though ACTH has actions throughout. Finally, the cells of the cortex originate from a stem cell population in the outer cortex or capsule, and migrate centripetally, changing their phenotype as they progress through the zones. Recent progress in understanding the development of the gland and the distribution of steroidogenic enzymes, trophic hormone receptors, and other factors suggests that this model needs refinement. Firstly, proliferation can take place throughout the gland, and although the stem cells are certainly located in the periphery, zonal replenishment can take place within zones. Perhaps more importantly, neither the distribution of enzymes nor receptors suggest that the individual zones are necessarily autonomous in their production of steroid. This is particularly true of the glomerulosa, which does not seem to have the full suite of enzymes required for aldosterone biosynthesis. Nor, in the rat anyway, does it express MC2R to account for the response of aldosterone to ACTH. It is known that in development, recruitment of stem cells is stimulated by signals from within the glomerulosa. Furthermore, throughout the cortex local regulatory factors, including cytokines, catecholamines and the tissue renin-angiotensin system, modify and refine the effects of the systemic trophic factors. In these and other ways it more and more appears that the functions of the gland should be viewed as an integrated whole, greater than the sum of its component parts.

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.

In the absence of hormone, corticosteroid receptors are primarily located in the cytoplasm, and they rapidly accumulate in the nucleus (t0.5 = 5 min) upon ligand binding. It is generally believed that the dissociation of hsp90 from the receptor is an absolute requirement for allowing its nuclear translocation. However, recent evidence suggests that hsp90 may remain associated with the glucocorticoid receptor during this process, and thus, the receptor nuclear localization signal (NLS) is not obscured by its presence. To determine the requirements for mineralocorticoid receptor (MR) nuclear transport, it was first shown that in rat kidney collecting duct cells, nuclear localization of MR in the presence of aldosterone was complete in 10 min. Although the hsp90 inhibitor radicicol delayed nuclear translocation, it did not prevent complete nuclear accumulation of MR at longer incubation times (t0.5 = 30-40 min). MR carbamylation generates a non-steroid-transformed receptor that, in contrast to native MR, is very stable in cell-free systems. In contrast to the full nuclear translocation of aldosterone-transformed MR, only a fraction of the carbamylated MR became nuclear in digitonin-permeabilized cells even though its NLS is exposed. Furthermore, while preincubation of permeabilized cells with NL1 peptide or anti-NL1 antibody fully inhibited the nuclear translocation of NL1-tagged albumin, neither treatment fully inhibited MR nuclear translocation. We postulate that there are at least two possible mechanisms for MR nuclear translocation. One of them is hsp90- and NL1-dependent, and the other functions in a manner that is independent of the classical pathway. Fil: Piwien Pilipuk, Graciela. Fundación Instituto Leloir; Argentina Fil: Vinson, Gavin P.. University Of London; Reino Unido Fil: Gomez Sanchez, Celso. University Of Mississippi; Estados Unidos Fil: Galigniana, Mario Daniel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Fundación Instituto Leloir; Argentina

Summary Syndrome X, the Metabolic Syndrome, and type II diabetes are closely related diseases that share risk factors and symptoms, notably insulin resistance. Several factors have been proposed either to mediate the disease(s) or to be their causes, and most converge on the endocrine/paracrine functions of the adipocyte. A common feature of such systems is their relative autonomy from systemic negative feedback regulation, for example by the HPA axis. We draw particular attention to two such mechanisms, both of which are associated with, and can cause, insulin resistance: the extra-adrenal production of corticosteroids, and the tissue renin angiotensin system of the adipocyte. These show another feature: the inter-regulation of glucocorticoid action and the RAS by positive feedback. Cortisol enhances the expression of 11β-HSD 1, and also of angiotensinogen and angiotensin type 1 receptors. In turn, angiotensin can stimulate further corticosteroid production, from the adrenal and perhaps from extra-adrenal sources. The instability inherent in such positive loops could account for the progressive nature of the disease(s), suggesting ways to break the circle.

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.

4-Hydroxy tamoxifen (OHT) and trilostane interact differently with the oestrogen receptor (ER). OHT is a competitive inhibitor whereas trilostane has direct, but non-competitive effects on ER. This study compared the effects of OHT and trilostane, in the presence of 17beta-oestradiol (E2) on gene expression in MCF-7 breast cancer cells using microarrays each representing nearly 20,000 human genes. Striking differences between the sets of genes affected by these two drugs were observed. Both OHT and trilostane affected transcription of genes involved in cell cycle regulation, cell adhesion and matrix formation, however, only 12.5% of trilostane down-regulated genes and 9.2% of up-regulated genes were similarly regulated by OHT. A selective up-regulation of ERbeta by trilostane, but not OHT, was observed and confirmed by qRT-PCR. Similar up-regulation of this gene by trilostane was observed in the uterus of trilostane-treated (4 mg/kg for 7 days) rats, in which ERbeta mRNA (3-fold) and ERbeta protein expression (10-fold) were both increased. These data show that OHT and trilostane regulate the expression of different sets of genes, reflecting their different modes of interaction with ER. Trilostane-specific up-regulation of ERbeta could explain its positive benefit rates in acquired tamoxifen resistance.

Angiotensin II has mitogenic and angiogenic effects and its receptors are widespread, particularly in epithelial tissue. Tissue renin angiotensin systems (tRASs) may be a local source of angiotensin II that has specific paracrine functions. To investigate the presence of a tRAS in normal human breast and tumours. Immunocytochemistry, and quantitative RT–PCR was used to establish: (i) the presence and localisation of RAS components, (ii) the possibility of their involvement in cancer. (1) mRNA coding for angiotensinogen, prorenin, angiotensin converting enzyme (ACE), and both AT1 and AT2 receptors was demonstrated in normal and diseased breast tissues. (2) (pro)renin was identified in epithelial cells in both normal and diseased tissue, but in invasive carcinoma, its distribution was mostly confined to fibroblasts or could not be detected at all. (3) Angiotensin converting enzyme was shown in epithelial cells in both normal and malignant tissue. The results are consistent with the hypothesis that a tRAS is present in the breast, and is disrupted in invasive cancer.

The adrenal inner zone antigen (IZA), which reacts specifically with a monoclonal antibody raised against the fasciculata and reticularis zones of the rat adrenal, was previously found to be identical with a protein variously named 25-Dx and membrane-associated progesterone receptor. IZA was purified as a glutathione S-transferase-fused or His6-fused protein, and its molecular properties were studied. The UV-visible absorption and EPR spectra of the purified protein showed that IZA bound a heme chromophore in high-spin type. Analysis of the heme indicated that it is of the b type. Site-directed mutagenesis studies were performed to identify the amino-acid residues that bind the heme to the protein. The results suggest that two Tyr residues, Tyr107 and Tyr113, and a peptide stretch, D99–K102, were important for anchoring the heme into a hydrophobic pocket. The effect of IZA on the steroid 21-hydroxylation reaction was investigated in COS-7 cell expression systems. The results suggest that the coexistence of IZA with CYP21 enhances 21-hydroxylase activity.

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.

Whole-genome assembly is now used routinely to obtain high-quality draft sequence for the genomes of species with low levels of polymorphism. However, genome assembly remains extremely challenging for highly polymorphic species. The difficulty arises because two divergent haplotypes are sequenced together, making it difficult to distinguish alleles at the same locus from paralogs at different loci. We present here a method for assembling highly polymorphic diploid genomes that involves assembling the two haplotypes separately and then merging them to obtain a reference sequence. Our method was developed to assemble the genome of the sea squirt Ciona savignyi, which was sequenced to a depth of 12.7× from a single wild individual. By comparing finished clones of the two haplotypes we determined that the sequenced individual had an extremely high heterozygosity rate, averaging 4.6% with significant regional variation and rearrangements at all physical scales. Applied to these data, our method produced a reference assembly covering 157 Mb, with N50 contig and scaffold sizes of 47 kb and 989 kb, respectively. Alignment of ESTs indicates that 88% of loci are present at least once and 81% exactly once in the reference assembly. Our method represented loci in a single copy more reliably and achieved greater contiguity than a conventional whole-genome assembly method.

After traumatic brain injury, progesterone has important neuroprotective effects in the nervous system. There is better functional outcome and less oedema formation in pseudopregnant rat females (high levels of endogenous progesterone) than in males. In addition to intracellular progesterone receptors, membrane binding sites of the hormone such as 25-Dx may also be involved in neuroprotection. In the present study we investigated the distribution of the membrane-associated progesterone-binding protein 25-Dx in rat brain. Immunohistochemical analysis showed that 25-Dx is particularly abundant in the hypothalamic area, circumventricular organs, and ependymal cells of the lateral walls of the third and lateral ventricles. A strong signal was also detected in the meninges. Double immunofluorescence immunolabelling and confocal microscopy showed that 25-Dx is co-expressed with vasopressin in neurones of the paraventricular, supraoptic and retrochiasmatic nuclei. Levels of 25-Dx expression were higher in pseudopregnant females than in males. After traumatic brain injury, 25-Dx expression was up-regulated in neurones and induced in astrocytes, which play an important role in regulating water and ion homeostasis. The expression of 25-Dx in structures involved in CSF production (choroid plexus) and in osmoregulation (circumventricular organs, hypothalamus and meninges), and its up-regulation after brain damage, point to a novel and potentially important role of this progesterone-binding protein in the maintenance of water homeostasis after traumatic brain injury.

The renin angiotensin system has been shown to have tissue-related functions that are distinct from its systemic roles. We showed that angiotensin II type 1 (AT1) receptors are present in mammalian sperm, and angiotensin II stimulates sperm motility and capacitation. In addition, angiotensin II is present in human seminal plasma at concentrations higher than found in blood. In testing the possibility that the prostate may be the source of seminal plasma angiotensin II, mRNA coding for angiotensinogen, (pro)renin, and angiotensin-converting enzyme were identified by RT-PCR in rat and human prostate and in prostate LNCaP cells, as well as the angiotensin receptors types 1 and 2 (AT1 and AT2) in human tissues and AT1 in rat. In human tissue, immunocytochemistry showed cellular colocalization of renin with the AT1 receptor in secretory epithelial cells. Confirmation of the capacity of the prostate to secrete angiotensin II was shown by the detection of immunoreactive angiotensin in media removed from rat prostate organ cultures and LNCaP cells. Rat prostate angiotensin secretion was enhanced by dihydrotestosterone, but LNCaP angiotensin was stimulated by estradiol. This stimulation was blocked by tamoxifen. Rat prostate AT1 receptor expression was much greater in prepuberal than in postpuberal rats but was not affected by a low-sodium diet. It was, however, significantly enhanced by captopril pretreatment. These findings all suggest the independence of prostate and systemic renin angiotensin system regulation. The data presented here suggest that the prostate may be a source of the secreted angiotensin II found in seminal plasma.

The ENCyclopedia Of DNA Elements (ENCODE) Project aims to identify all functional elements in the human genome sequence. The pilot phase of the Project is focused on a specified 30 megabases (∼1%) of the human genome sequence and is organized as an international consortium of computational and laboratory-based scientists working to develop and apply high-throughput approaches for detecting all sequence elements that confer biological function. The results of this pilot phase will guide future efforts to analyze the entire human genome.

Since its discovery, it has been generally assumed that the primary function of the zona glomerulosa of the adrenal cortex is the secretion of aldosterone. Taking evidence from the rat, and recognising that there is probably considerable species variation, I argue here that the glomerulosa in fact has many functions, including aldosterone synthesis, but is probably only a relatively poor de novo source of steroid. In vitro, the CYP11B2 (aldosterone synthase) of the glomerulosa can and does utilise as substrates products arising from CYP11B1 (11beta-hydroxylase) activity in fasciculata cells. Whether it does in vivo is open to question, but corticosterone and 18-hydroxydeoxycorticosterone are both present in circulating rat plasma at suitable concentrations. Such a mechanism would explain several inconsistencies in the literature, including the anomalous distribution of steroidogenic enzymes in the glomerulosa, the stimulation of CYP11B1 products by aldosterone secretagogues such as potassium ions or angiotensin II, the partial dependence of aldosterone secretion in vivo on an intact pituitary, the sensitivity of aldosterone secretion to tissue disruption in vitro, and the "late pathway" regulation of aldosterone synthesis.

Progesterone provides neuroprotection after spinal cord injury, but the molecular mechanisms involved in this effect are not completely understood. In this work, expression of two binding proteins for progesterone was studied in intact and injured rat spinal cord: the classical intracellular progesterone receptor (PR) and 25-Dx, a recently discovered progesterone membrane binding site. RT-PCR was employed to determine their relative mRNA levels, whereas cellular localization and relative protein levels were investigated by immunocytochemistry. We observed that spinal cord PR mRNA was not up-regulated by estrogen in contrast to what is observed in many brain areas and in the uterus, but was abundant as it amounted to a third of that measured in the estradiol-stimulated uterus. In male rats with complete spinal cord transection, levels of PR mRNA were significantly decreased, while those of 25-Dx mRNA remained unchanged with respect to control animals. When spinal cord-injured animals received progesterone treatment during 72 h, PR mRNA levels were not affected and remained low, whereas 25-Dx mRNA levels were significantly increased. Immunostaining of PR showed its intracellular localization in both neurons and glial cells, whereas 25-Dx immunoreactivity was localized to cell membranes of dorsal horn and central canal neurons. As the two binding proteins for progesterone differ with respect to their response to lesion, their regulation by progesterone, their cellular and subcellular localizations, their functions may differ under normal and pathological conditions. These observations point to a novel and potentially important role of the progesterone binding protein 25-Dx after injury of the nervous system and suggest that the neuroprotective effects of progesterone may not necessarily be mediated by the classical progesterone receptor but may involve distinct membrane binding sites.

The 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 the beta1 integrin receptors.Pure 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 both beta1 integrin expression and tumour grade. Additionally, the effect of beta1 receptor blockade with monoclonal antibody (mAb) was assessed.Invasion was significantly greater in grade II than grade III tumour cells (p=0.0012). Anti-integrin beta1 monoclonal antibody inhibited cancer cell invasion by a mean of 83.96 +/- 4.80%.The invasion assay confirmed the fundamental importance of beta1 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 integrin beta1-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.

The clear morphological distinction between the cells of the different adrenocortical zones has attracted speculation and experiment to interpret their functions and the ways in which they are regulated. Considerable data have been produced in recent years that has benefited a fuller understanding of the processes of steroidogenesis and of cell proliferation at the molecular level. This now enables the reexamination of earlier concepts. It is evident that there is considerable species variation, and this article, dealing mainly with the rat, reaches conclusions that do not necessarily apply to other mammals. In the rat adrenal, however, the evidence suggests that the greatest differences between the functions of the zones are between the glomerulosa and the fasciculata. Here the sometimes all-or-nothing demarcation in their complement of components associated with steroidogenesis or with cell proliferation suggests a stark division of labor. In this model the fasciculata is the main engine of steroid hormone output and the glomerulosa is the site of cell proliferation, recruitment, and differentiation. Regulating these functions are angiotensin II and other paracrine components that modulate and maintain the glomerulosa, and ACTH, that maintains the fasciculata, and recruits new fasciculata cells by transformation of proliferating glomerulosa cells. Grafted onto this mostly vegetative function of the glomerulosa is CYP11B2, limited to just a fraction of the outer glomerulosa in rats on a normal laboratory diet and generating aldosterone (and 18-hydroxycorticosterone) from precursors whose origin is not, from the evidence summarized here, very clear, but may include the fasciculata, directly or indirectly. The biosynthesis of aldosterone in the rat certainly requires reinterpretation. Microsc. Res. Tech. 61:227–239, 2003. © 2003 Wiley-Liss, Inc.

Currently available antioestrogens, such as tamoxifen, are competitive inhibitors that bind to the ligand binding sites of oestrogen receptors, ERα and ERβ. The search for alternative anti-hormone therapies is prompted by the need for drugs that are effective when tumours become tamoxifen resistant. The existence of different receptor isoforms also raise the possibility of improving selectivity. Earlier use of the 3β-hydroxysteroid dehydrogenase inhibitor, trilostane (4α,5- epoxy-17β-hydroxy-3-oxo-5α-androstane-2α-carbonitrile), suggested that it had beneficial actions in breast cancer that were only partially attributable to inhibition of steroidogenesis. The present studies on the interactions of trilostane with oestrogen receptors show that it (i) inhibits oestrogen-stimulated proliferation in MCF-7 breast cancer cells, (ii) enhances the affinity of oestradiol binding to ER in rat uteri and specifically increases oestradiol binding to an ERβ-like isoform, (iii) inhibits ERα and ERβ binding to the classical vitellogenin gene oestrogen response element (ERE) and (iv) inhibits oestrogen-stimulated gene transcription in ERE-linked reporter systems in MCF-7 cells. The results demonstrate a novel, presumably allosteric, mode of antioestrogen action. The beneficial actions of trilostane in breast cancer may be attributed to the combination of this antioestrogen effect with its well documented suppression of steroidogenesis. © 2002 Wiley-Liss, Inc.

Vasoactive intestinal peptide (VIP) and Neuropeptide Y (NPY) are localised in the capsule and zona glomerulosa region of the adrenal cortex, where they play an important role in regulating steroidogenesis and adrenal blood flow. This study investigates the effect of these neuropeptides on adrenocortical cellular proliferation and steroidogenesis in vitro. Capsular/glomerulosa and innerzone/medulla preparations were either stimulated acutely with NPY or VIP (both 10(-6) M) for up to 2 hours or for 24 hours, four and eight days in vitro in eagles MEM (3.4 mM K+). DNA synthesis was determined using immunocytochemistry through the incorporation of the thymidine analogue 5-bromo-2'-deoxyridine (BrdU, 20 mg/mL). Phosphorylation of mitogen activated protein kinase ERK1/2 was assessed by western blotting. Both VIP (10(-6) M) and NPY (10(-6) M) treatment caused an increase in DNA synthesis after four days in culture. Acute NPY treatment caused an increase in ERK1 and 2 phosphorylation (p0.01) in the capsular/zona glomerulosa. Vasoactive intestinal peptide treatment caused a significant increase in ERK 1/2 phosphorylation (p0.05) only in innerzones/medulla preparations. Both responses were maximal between 10 and 30 min of incubation and decrease thereafter. These data provide further evidence for the role of the mitogen activated protein kinases ERK1 and 2 in the proliferative events in the adrenal gland and demonstrate stimulation of cell division by the adrenal neuropeptides VIP and NPY in vitro.

Using immunological methods, a protein specific to the inner zones of the rat adrenal cortex, and called inner zone antigen (IZAg), was previously shown to have two interrelated forms of 26 kDa (IZAg1) and 55–60 kDa (IZAg2), and to have an action on steroid hydroxylation. After two-dimensional gel electrophoresis, and immunoaffinity column purification, N-terminal amino-acid analysis showed that the first 12 amino acids were identical to those of a recently described putative membrane located progesterone receptor (PPMR). RT-PCR was then used to generate the cDNA of this protein, using RNA extracted from rat adrenals. A glutathione S-transferase (GST)-fusion construct was expressed in Escherichia coli, and shown to generate an immunoreactive product of molecular mass consistent with its identification as IZAg1. More detailed examination of the distribution of this protein, not only in the zona fasciculata/reticularis of the adrenal cortex, but also in the Leydig cell, kidney and liver, suggest it may have a role in steroid hormone synthesis and/or metabolism.

Fertilization-promoting peptide (FPP) regulates the adenylyl cyclase (AC)/cAMP pathway to elicit capacitation-dependent responses, stimulating capacitation in uncapacitated spermatozoa and then arresting it in capacitated cells, thereby inhibiting spontaneous acrosome reactions. Like FPP, calcitonin and angiotensin II are found in seminal plasma and so might affect sperm function; this study investigated responses in uncapacitated and capacitated mouse spermatozoa to these three peptides. Both calcitonin (5 ng/ml) and angiotensin II (1 and 10nmol/l), like FPP (100nmol/l), significantly stimulated capacitation, assessed using chlortetracycline (CTC) fluorescence and fertilization in vitro analyses. Combinations of two or three peptides, at high and low, non-stimulatory concentrations, were more stimulatory than the individual peptides, suggesting that they may act on the same signalling pathway, plausibly AC/cAMP; preliminary data indicate that calcitonin does stimulate cAMP production. In capacitated cells, FPP and calcitonin elicited pertussis toxin-sensitive inhibition of spontaneous acrosome loss, suggesting involvement of inhibitory G proteins; angiotensin II had no detectable effect. When all three peptides were used, angiotensin II did not interfere with inhibitory responses to FPP/calcitonin. These results suggest that angiotensin II, calcitonin and FPP may somehow modulate the AC/cAMP signal transduction pathway, but the precise mechanisms involved have yet to be elucidated.

Aims: Integrins are a major family of cell adhesion molecules whose function is perturbed in tumour invasion and metastasis. Angiotensin II (A II) is well-known in the systemic control of water and electrolyte homeostasis and haemodynamics, but recent evidence points to an additional local renin–angiotensin system (RAS) with possible long-term trophic effects including carcinogenesis. Methods: The effect of angiotensin II on MCF-7 human breast cancer cell line integrin expression was evaluated with immunocytochemistry (ICC) and immunoprecipitation (IP). Results: The experiments demonstrated a 1.40±0.14-fold increase in bgr; 1 integrin expression on MCF-7 cells following treatment with A II. Conclusions: These findings report the first evidence of an association between integrins and the RAS in human breast cancer cells and suggest a novel research avenue for future anti-metastatic strategies, through the manipulation of cell adhesion mechanics, in the management of invasive human breast cancer.

Stimulation of aldosterone biosynthesis by angiotensin II (AII) is thought to be mediated via the PLC, IP3 and intracellular calcium signalling pathway. MAPK (p42/p44) is involved in cell proliferation, and is also activated by AII, but its role in the adrenal response to dietary sodium is unclear. To study the relationship between AII receptor (ATR), MAPK and PKC isoforms, PKCalpha and PKCepsilon, mature Wistar rats were maintained on low or high sodium diets for 1 week. In adrenals from animals on a sodium deplete diet, total ligand binding to both ATR subtypes decreased in the zona glomerulosa (ZG). Under these conditions, active MAPK in the ZG decreased paralleling a decrease in active PKCalpha. In the inner zones (IZ), largely reflecting medullary events, low sodium did not affect MAPK activity. However active PKCalpha decreased. In adrenals from sodium-loaded animals, type 2 ATR (AT2R) binding was reduced in the ZG, while type 1 ATR (AT1R) increased in the IZ. Active MAPK increased in ZG, as did active PKCalpha and PKCepsilon. In IZ, ERK, PKCalpha and PKCepsilon were unchanged. These results suggest that in the ZG and IZ, two different modes of MAPK regulation may exist, utilising different PKC isoforms.

All the components of the renin-angiotensin system (RAS), including the AT(1)receptor, have previously been shown to be present in the human term placenta. However, the presence of the RAS components has not been fully investigated in the human placenta throughout pregnancy. The aim of this study was to examine the localization of the angiotensin receptors AT(1)and AT(2)using immunocytochemistry and the expression of prorenin, angiotensinogen and the AT(1)and AT(2)receptor mRNA using RT-PCR in the human placenta in the first, second and third trimesters of pregnancy. Localization of the AT(1)receptor was shown throughout gestation in the syncytiotrophoblast, cytotrophoblast, Hofbauer cells and the fetal vascular endothelium. Expression of mRNA for prorenin, angiotensinogen and the AT(1)receptor was shown in the placenta throughout gestation. However, localization or mRNA expression of the AT(2)receptor was not detected in any of the placental samples studied. These results clearly show the expression of a majority of the components of the RAS in the placenta from early gestation onwards. Therefore, these results suggest that the RAS may have a role in the human placenta throughout gestation.

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 effects of angiotensin II (ANG II), the angiotensin type 1 (AT1) receptor antagonist losartan, and low-sodium diet on rat adrenal cell proliferation were studied in vivo with immunocytochemistry. Both ANG II and low-sodium diet increased proliferation of endothelial cells of the zona glomerulosa. Losartan prevented ANG II-induced hyperplasia of glomerulosa cells but not the effects of a low-sodium diet. Glomerulosa cells after ANG II + losartan treatment appeared hypertrophied compared with those of controls. Proliferative effects of ANG II and low-sodium diet in the reticularis were blocked by losartan. No changes were seen in the fasciculata. Proliferation in the medulla was increased with losartan, was decreased by ANG II, but was unaffected by low-sodium diet. In conclusion, 1) cell hypertrophy and proliferation of glomerulosa cells are mediated by AT1 receptor-dependent and -independent processes, 2) proliferation of reticularis cells is controlled by AT1 receptors, and 3) reciprocal control of chromaffin cell proliferation by ANG II may involve indirect AT1-dependent processes.

1. Although much work has addressed the functional significance of mammalian adrenocortical zonation, less attention has been paid to its developmental origins and the factors that maintain it. Recent concepts of tissue differentiation hold that cells respond to local morphogenic stimuli that are generated in a paracrine manner. 2. In fact, the adrenal cortex represents an ideal mammalian in vivo model for such studies: few others exist. While several components may contribute to the establishment of a developmental polarity in the gland, including products of capsular and neural elements, compelling evidence now suggests that the tissue renin-angiotensin system (RAS) has a critical role. 3. We have examined the roles of these and other paracrine morphogens and growth factors and of specific transcription factors in adrenocortical cellular proliferation and development. From data obtained by using in situ hybridization to determine their cellular location, we propose a hierarchy of potential tissue modelling agents. These include morphogens, such as angiotensin II derived from the intra-adrenal RAS, growth factors (e.g. basic fibroblast growth factor), which can be considered to be the paracrine amplifiers of the morphogenic signal, and, finally, transcription factors, such as C-fos, that directly stimulate mitosis and other events of differentiation.

The angiotensin II type 1 (AT1) receptor is present in a wide variety of human and animal tissues, and is particularly abundant in epithelial cells. Because of this, and because it is known that tissue renin angiotensin systems (RASs) exist that have specific local functions, we investigated the expression and localisation of components of the RAS in normal and diseased breast tissue. Using a monoclonal antibody to the AT1 receptor, immunocytochemistry confirmed that the AT1 receptor was characteristically distributed in ductal epithelial cells in both normal and malignant tissue, and in most, although not all, cells in invasive tumours. Transcription of prorenin mRNA was studied by in situ hybridisation, using a DIG-ddUTP tail-labelled probe specific for the human prorenin gene. In normal tissue, and in cases of ductal carcinoma in situ, prorenin mRNA was distributed in myoepithelial cells and in a band of connective tissue cells completely surrounding the AT1-containing ductal epithelial cells. This prorenin transcribing tissue was disrupted and attenuated in invasive tumours, and in some of these, prorenin mRNA transcription could not be detected at all. Functions ascribed to the tissue RASs include regulation of mitosis and tissue modelling, as well as fluid and electrolyte transport. The results presented here strongly suggest the possibility that a tissue RAS may also be present in the breast, closely coupled to the provision of angiotensin II to the AT1 receptors in ductal epithelial cells. This mechanism is disrupted in cancer.

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.

The stimulation of the zona glomerulosa phenotype by a low sodium diet is characterised by increased expression of aldosterone synthase accompanied by increases in (pro)renin, bFGF, c-fos and c-jun gene transcription. In contrast ACTH diminishes the specific glomerulosa phenotype. The EGF related transmembrane protein preadipocyte factor 1 (Pref-1) is specifically found in the zona glomerulosa and medulla of the adult rat adrenal as well as being expressed in fetal tissue. In addition the orphan nuclear receptor SF-1/Ad4bp considered to be an important factor in fetal adrenocortical development and differentiation may also have a role in zonal differentiation in the adult. To investigate their roles, adrenals were taken from adult Wistar rats maintained on a low sodium diet or ACTH treated (enhancing or diminishing zona glomerulosa function respectively) and compared with untreated controls. Localisation of Pref-1 was carried out by immunocytochemistry using the polyclonal antibody ZOG (anti Pref-1) and of SF-1 using a rabbit antiserum to SF-1. The experimental treatment resulted in a decrease and increase in Pref-1 expression in ACTH treated and low sodium diet treated rats respectively, in accordance with changes in abundance of glomerulosa cells. SF-1 expression was expected throughout the adrenal cortex, without zonal differentiation, though somewhat decreased by ACTH treatment. Of these two factors, only Pref-1 can be considered to have a role in zonal differentiation.

The continued acquisition of information on the distribution of expression of components of the renin-angiotensin system shows that it has functions in the tissues that are quite unrelated to its systemic actions. In particular, both type 1 and type 2 angiotensin receptors are found in many tissues of the reproductive system of both sexes. In addition, the widespread occurrence of (pro)renin, angiotensin converting enzyme and angiotensinogen suggests that the generation of angiotensin II within the tissues occurs at sites close to its sites of action. The data suggest that angiotensin II operates as an important paracrine agent with profoundly significant roles in several functions of the reproductive system, and in fertility.

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

Increasing evidence suggests that the actions of classical stimulants of adrenocortical growth and function, such as ACTH or dietary sodium restriction, may partially be mediated via locally produced regulators. Several peptide growth factors, such as basic fibroblast growth factor, insulin-like growth factors, and transforming growth factor-beta 1, have emerged in recent years as multifunctional molecules that typically play such regulatory roles. Adrenocortical cells are highly responsive to these growth factors, in particular in the regulation of cell growth and differentiated functions, such as steroidogenesis. In addition, growth factor expression in the adrenal cortex has been shown to be regulated by physiological stimulants. The spatial expression, release, and activation of these growth factors may, therefore, locally mediate or amplify the actions of the hypothalamo-pituitary axis and the renin-angiotensin system on adrenocortical proliferation, differentiation, and steroidogenesis.

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 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 mediating the response of the tissue to stimulation by ACTH and potassium ions, but its role in the response to angiotensin II stimulation has not been addressed. To examine this, rat 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. The results illustrate the importance of the tissue RAS in the synthesis of aldosterone and the response to angiotensin II, and the two sets of data suggest that the fasciculata and glomerulosa zones interact in the formation of aldosterone. One way in which this may occur is by the mobilisation of fasciculata synthesised substrate, such as 18-hydroxydeoxycorticosterone (18-OH-DOC), for conversion by the glomerulosa, which is apparently supported by endogenous angiotensin II.

Using an antibody (6313/G2) directed against a specific sequence in the extracellular domain of the type 1 angiotensin II receptor (AT1), we demonstrated the presence of angiotensin II (AII) receptors in human fallopian tube. Immunoperoxidase staining for AT1 receptor showed positive staining in the epithelium of the tubal mucosa. The intensity of staining varied depending upon the hormonal status at the time of salpingectomy, being strongest in the proliferative phase of the ovarian cycle and weakest after menopause. Ligand binding assay confirmed that the AII receptor concentration was highest in the mucosa of fallopian tubes from premenopausal women. Mucosa from the ampullary segment had higher concentrations of AII receptor than the fimbrial and isthmic segments in both premenopausal and postmenopausal women. Displacement studies using specific AII receptor subtype antagonists showed that approximately 60% of the total activity could be displaced by CGP42112B (type 2 specific) and 40% by losartan (AT1 specific). Immunoblotting confirmed that the antibody detected a protein of approximately 60 kDa. Functional studies showed that AII had a stimulatory action on tubal ciliary beat frequency, but had no significant effect on myosalpingseal activity. This effect was achieved at nanomolar concentrations of AII; further increases in the AII concentration were without additional effect. The stimulatory effect of AII was inhibited by the specific AT1 antagonist losartan, whereas the type 2 antagonist, CGP42112B, had no effect. The data demonstrate that AII may play an important role in ovum transport and fertility.

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.

The extracellular matrix consists of the interstitium and the basement membrane. Cellular interaction with fibronectin, laminin and collagen provides a possible mechanism by which cancer cells adhere, invade and metastasize. The integrins are a major family of adhesion molecules that recognize epitopes on the extracellular matrix as ligands. These include the α2β1, α3β1, αvβ1 and αvβ5 integrins, most of which were found to be expressed on MCF-7, T47D, MDA-MB-231, ZR75-1 and Hs578T breast cancer cell lines. Each cell line adhered to the matrix proteins in a dose-dependent manner and was inhibited by monoclonal antibodies against relevant integrins. Only Hs578T was significantly invasive through fibronectin but both Hs578T and MDA-MB-231 invaded through laminin and type IV collagen in an in vitro assay. The invasive potential of these cell lines could be inhibited by integrin antibodies added to cells before incubation, but the addition of antibodies after cells were allowed to adhere to the matrix failed to inhibit invasion. Inhibition of cellular adhesion to the matrix reduced the invasive potential of breast cancer cell lines. As integrin antibodies inhibit cell invasion in vitro, the integrins may be of potential value as antitumour therapeutic agents.

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

Using a specific monoclonal antibody (6313/G2) to the first extracellular domain of the type 1 receptor (AT1), we showed that most of the receptor is internalised in the rat glomerulosa cell. When viable glomerulosa cells are incubated with 6313/G2. the receptor is transiently concentrated on the cell surface, and aldosterone output is stimulated. This stimulated output is enhanced by neither threshold nor maximal stimulatory concentrations of AII amide, although the antibody does not inhibit AII binding to the receptor. The antibody directly stimulates inositol trisphosphate (IP3) generation, but, while having no intrinsic action on protein kinase C (PKC) activation, it significantly inhibits the PKC response to angiotensin II.The data suggest that although the receptor is mostly internalized, recycling to the plasma membrane is constitutive, or regulated by unknown factors. Retention of the AT1 receptor in the membrane is alone enough to allow sufficient G protein interaction to generate maximal steroid...

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