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S Marsigliante, A Muscella, G P Vinson, and C Storelli


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.

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M Montiel, S Barker, G P Vinson, and E Jiménez


The angiotensin II (Ang II)-binding sites in rat adrenal gland membranes were characterized using 125I-radiolabelled Ang II. While Scatchard analysis identified a single population of Ang II receptor sites, isoelectric focusing (IEF) on polyacrylamide gels revealed four peaks of specific Ang II binding which migrated to isoelectric points (pI values) 6·8, 6·7, 6·5 and 6·3. In binding assays in the presence of an excess of the Ang II receptor AT1 subtype antagonist DuP 753, a monophasic dose-dependent displacement of 125I-labelled Ang II binding by the Ang II receptor AT2 subtype antagonist CGP42112A was observed, and vice versa. In this system, reduction of disulphide bridges using 1 mmol dithiothreitol (DTT)/l markedly increased the number of binding sites in the adrenal zona glomerulosa without affecting receptor affinity.

Using IEF, it was found that both DuP 753 and CGP42112A were able to reduce specific binding of each of the four peaks to some extent. However, the predominant effect of DuP 753 was to reduce the labelling of the isoform at pI 6·7 substantially, while CGP42112A significantly inhibited the specific 125I-labelled Ang II binding to the pI 6·3 isoform. When DuP 753 and CGP42112A were used together, specific binding of 125I-labelled Ang II to the isoforms of pI values 6·8, 6·7 and 6·3 was completely eliminated. These data suggest that the four peaks of specific binding found may be composed of different isoforms of both AT1 and AT2 receptor subtypes and that the Ang II receptor isoforms which migrated to pI 6·7 and pI 6·3 are predominantly composed of AT1 and AT2 receptor subtypes respectively. Interestingly, in the presence of both antagonists, 8·7 ± 0·9% of the specific binding migrating at pI 6·5 remained unaffected. This finding suggests the presence of an additional subtype, which is neither AT1 nor AT2, in the rat adrenal zona glomerulosa.

In further studies, pretreatment with DTT was found to increase the specific 125I-labelled Ang II binding of all four isoforms. Moreover, DTT also produced a further specific binding component between pI 6·5 and pI 6·7 which exhibited AT2 subtype pharmacology in DTT-treated preparations. Since DTT has been reported to enhance only AT2 subtype binding this also suggests that the different isoforms may contain components related to both AT1 and AT2 receptor subtypes.

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S. Kapas, C. D. Orford, S. Barker, G. P. Vinson, and J. P. Hinson


The intracellular mechanisms of action of α-MSH in rat adrenocortical cells were examined. When rat adrenal capsule (largely glomerulosa) cells were stimulated with a range of concentrations of α-MSH there was significant stimulation of aldosterone secretion at 10-10 mol/l, although cyclic AMP was not increased until high concentrations of α-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/1.

When rat adrenal whole capsules were incubated with α-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 α-MSH there was significant generation of [3H]inositol trisphosphate (IP3) at concentrations of α-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 α-MSH.

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S. Barker, S. M. Laird, M. M. Ho, G. P. Vinson, and J. P. Hinson


We have previously reported the production of a monoclonal antibody (IZAb) which interacts with an antigen, found predominantly in rat adrenal inner zone tissue, which may have a role in steroidogenesis. Here we describe initial studies on its characterization.

Immunoblot analysis of rat adrenocortical proteins obtained from fresh tissue and separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, showed that the IZAb interacted with a protein with a molecular mass of approximately 30 000 Da (IZAg1). This protein was found predominantly in rat adrenal inner zone tissue. Small amounts were seen in the zona glomerulosa, while no corresponding protein was seen in rat ovary, heart, liver, testis or kidney tissue. Subcellular fractionation of rat adrenocortical inner zone tissue and immunoblot analysis showed that the IZAg1 was present in the microsomal and mitochondrial fractions of the cell, but was absent from the cytosol. Invivo treatment with ACTH (100 μg/day) for more than 5 days also increased the expression of this protein by rat adrenal inner zone tissue, and this was coincident with increased corticosterone and 18-hydroxydeoxycorticosterone (18-OH-DOC) production in incubations of inner zone tissue in vitro.

In experiments involving the short-term culture of rat adrenal inner zone cells, IZAb interacted with two protein bands. IZAg1 was detected as a minor band in untreated control cells, while another protein with a molecular mass of approximately 60 000 Da, designated IZAg2, was present in greater amounts. Treatment of cells for 48 h with either ACTH (1 μmol/l) or dibutyryl-cAMP (100 μmol/l) resulted in apparent increased expression of IZAg1 and diminished levels of IZAg2. As in the in-vivo treatments, the increase in IZAg1 was associated with a corresponding increase in corticosterone and 18-OH-DOC production.

These findings suggest that the IZAb recognizes a protein (IZAg2) which occurs in unstimulated adrenal cells. On stimulation by steroidogenic agents, this protein becomes processed to yield a smaller protein (IZAg1) which is associated with enhanced adrenal steroidogenesis.

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E. Jimenez, S. Marsigliante, S. Barker, J. P. Hinson, and G. P. Vinson


Angiotensin II (AII) receptors were identified in rat tissue membranes by specific binding of 125I-labelled AII. Using an isoelectric focusing technique, two forms of the high-affinity AII receptor were identified in rat adrenal zona glomerulosa and liver membranes. These migrated to isoelectric points (pI) 6.8 and 6.7. Two low-affinity forms migrated to pI 6.5 and 6.3. The two high-affinity forms were in greatest abundance in the zona glomerulosa, while the low-affinity pI 6.5 isoform was predominant in liver membranes. In uterine membranes both low-affinity isoforms were observed, but there was only one of the high-affinity forms (pI 6.7).

Concentrations of AII receptor isoforms were increased in the zona glomerulosa of sodium-deprived rats.

Reduction of disulphide bridges with dithiothreitol (DTT) had different effects on the various AII receptor isoforms. Thus 1 mmol DTT/l caused a twofold increase in 125I-labelled AII binding in zona glomerulosa membranes. DTT produced no appreciable differences in specific AII binding in uterine membranes, whereas there was a 50% reduction of binding in liver membranes. At 20mmol/1, DTT greatly decreased AII binding in all tissues.

The data suggest the existence of multiple forms of AII receptors which may have different functions.

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S. M. Laird, J. P. Hinson, G. P. Vinson, N. Mallick, S. Kapas, and R. Teja


The involvement of the calcium messenger system in the control of steroidogenesis in the rat and bovine adrenal cortex has been studied extensively. However the role of these second messengers in the control of human adrenocortical function is not established. This was therefore studied by incubating collagenase-dispersed human adrenocortical cells with the calcium ionophore A23187 and the protein kinase C activator phorbol 12-myristate 13-acetate (TPA). The effects of the calcium channel blocker verapamil on basal and stimulated steroidogenesis were also studied.

Both TPA (1 pmol/l–10 μmol/l) and A23187 (1 nmol/l–10 μmol/l) caused a dose-dependent increase in cortisol, aldosterone and corticosterone production. Verapamil (10 μmol/l) inhibited the increase in aldosterone, corticosterone and cortisol produced in response to ACTH(1–24), potassium, and desacetyl-αMSH. Unlike previous results in the rat, these effects were not specific for aldosterone secretion.

The results suggest that, as in other species, calcium mobilization and protein kinase C activation have a role in the control of steroidogenesis in the human adrenal cortex. However, in contrast to the rat, these mechanisms appear to be involved in the control of steroidogenesis in both the zona glomerulosa and inner zone cells.

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G. P. Vinson, S. M. Laird, J. P. Hinson, N. Mallick, S. Marsigliante, and R. Teja


When rat adrenal whole capsules, containing the zona glomerulosa, were incubated, addition of the protein kinase C inhibitors TMB-8 (10 μmol/l), W7, H7, polymyxin-B and sphingosine (all 1 μmol/l) was found to inhibit the steroidogenic response to trypsin. Aldosterone and 18-hydroxycorticosterone were strongly, and corticosterone moderately, affected, while the production of 18-hydroxydeoxycorticosterone was neither stimulated by trypsin nor inhibited by the protein kinase C inhibitors. Addition of neomycin, which prevents substrate interaction with phospholipase C, also inhibited the response to trypsin, while addition of phospholipase C itself stimulated aldosterone, 18-hydroxycorticosterone and corticosterone production with the same tissue sensitivity as trypsin. Addition of phospholipase A2 had no effect. Direct assay of protein kinase C activity showed that trypsin stimulation effected the translocation of Ca2+/phospholipid-activated protein kinase C from the cytosolic to the membrane fraction. When glomerulosa tissue was incubated with [32P]ATP, and cytosolic proteins were subjected to isoelectric focusing on polyacrylimide gels, autoradiography showed that incorporation of 32P into several protein components was increased by trypsin stimulation.

It was concluded that trypsin exerts its stimulatory effects on steroidogenesis by activating protein kinase C; not, however, by generating the Ca2+/phospholipid-independent fragment, but possibly by enhancing the activity of phospholipase C.

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S Kapas, A Purbrick, S Barker, G P Vinson, and J P Hinson


It is well established that ACTH and angiotensin II (Ang II) stimulate aldosterone secretion from rat adrenal zona glomerulosa cells in vitro and mediate their steroidogenic effects via the cyclic AMP (cAMP) pathway and phosphoinositide turnover respectively. α-MSH also stimulates aldosterone secretion from zona glomerulosa cells in vitro, and recent studies from our laboratory have shown that its steroidogenic effects are mediated by increases in inositol 1,4,5-trisphosphate (IP3) production. α-MSH also stimulates adenylyl cyclase activity, but only at concentrations that are supramaximal for stimulation of steroidogenesis. The observation that α-MSH-stimulated IP3 accumulation declines as the activity of adenylyl cyclase increases prompted further studies on the interactions of cAMP and phosphoinositide production.

The effects of α-MSH and ACTH on Ang II-stimulated steroidogenesis and IP3 accumulation were studied. On addition of increasing concentrations of ACTH, both the aldosterone and IP3 responses to Ang II were significantly inhibited; however, only high concentrations of α-MSH achieved this effect. These results suggest that cAMP or a cAMP-dependent event is able to inhibit phospholipase C activity. This hypothesis was tested by measuring IP3 production in Ang II-stimulated zona glomerulosa cells exposed to two different concentrations of α-MSH: 1 nmol/l, which stimulates the generation of IP3, and 1 μmol/l, which activates adenylyl cyclase. It was found that this high concentration of α-MSH significantly inhibited Ang II-stimulated aldosterone secretion and IP3 levels. In addition, α-MSH reduced 125I-labelled Ang II binding to rat adrenal zona glomerulosa cells. ACTH and cAMP also inhibited Ang II binding, thus supporting the hypothesis that cAMP (or a cAMP-mediated event) inhibits Ang II receptor function.

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G. P. Vinson, S. M. Laird, B. J. Whitehouse, and J. P. Hinson


Recent data have implicated the phosphatidylinositol/calcium second-messenger system in the control of aldosterone secretion by the adrenal zona glomerulosa. However, in the rat adrenal there are few reports of a direct effect of protein kinase C activation on steroid secretion, while the effects of calcium mobilization may be variable. Since the rat adrenal zona glomerulosa is sensitive to the mode of tissue preparation, these mechanisms were reinvestigated in intact (non-dispersed) capsular tissue and collagenase-dispersed zona glomerulosa cells.

Steroidogenesis in the intact zona glomerulosa was markedly affected by agonists of the calcium messenger system. Most notably, aldosterone and 18-hydroxycorticosterone (18-OH-B) secretion were stimulated by A23187 (100 nmol to 10 μmol/l) and BAY K 8644 (500 nmol/l). Phorbol 12-myristate 13-acetate (TPA; 1 pmol to 1 μmol/l) stimulated aldosterone secretion at all doses and caused a dose-dependent increase in 18-OH-B and 18-hydroxydeoxycorticosterone (18-OH-DOC) secretion. Corticosterone secretion was slightly increased in the presence of A23187 but not by TPA or BAY K 8644. Production of 18-OH-DOC was unaffected by A23187 and BAY K 8644. The calcium channel antagonist verapamil (10 μmol/l) inhibited ACTH-stimulated aldosterone secretion by the intact zona glomerulosa but had no effect on corticosterone secretion.

Verapamil (10 μmol/l) also inhibited the increase in aldosterone secretion by collagenase-dispersed zona glomerulosa cells stimulated by ACTH (100 fmol to 100 nmol/l), angiotensin II (100 pmol to 10 nmol/l) and potassium (5·9 and 8·4 mmol/l); stimulated corticosterone secretion was unaffected. Aldosterone secretion by dispersed zona glomerulosa cells was unaffected by the calcium ionophore A23187 (10 nmol to 100 μmol/l) or by TPA (1 nmol to 10 μmol/l). Corticosterone secretion was also unaffected by A23187 over the same dose range, but was increased slightly by high doses of TPA (10 and 1 μmol/l), while the calcium channel agonist BAY K 8644 had no effect on either steroid.

The results show that calcium mobilization and protein kinase C activation can stimulate steroid secretion in the rat adrenal zona glomerulosa and that large and reproducible effects are seen when intact tissue is used. In general only 18-OH-B and aldosterone secretion were affected; effects on corticosterone and 18-OH-DOC were much less marked. Together with the effects of calcium blockers, the data strongly support the view that protein kinase C activation and calcium mobilization are primarily involved in the control of the 'late pathway' of aldosterone biosynthesis.

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S. Marsigliante, V. A. Baker, J. Puddefoot, S. Barker, and G. P. Vinson


The variability in the profile of oestrogen receptor (ER) isoforms in breast tumours has been studied.

Using low-resolution isoelectric focussing (IEF), two major ER isoforms with isoelectric point (pI) values of 6.1 and 6.6 could be identified, with corresponding sedimentation coefficients in sucrose density gradients of 8 S and 4 S respectively.

Using high-resolution IEF or immunoblotting, the pI 6.6 form (4S) was shown to be composed of three different species, with pI values of 6.3, 6.6 and 6.8, while the oligomeric pI 6.1 protein (8 S) did not show charge heterogeneity. Data were obtained on the soluble receptors from supernatants of 42 ER-positive primary breast tumour homogenates using high-resolution IEF to obtain ER isoform profiles. It was found that 54.7% of tumours contained the isoforms at pI 6.6 and 6.1, while only 11.9% contained the full complement of isoforms (pI 6.1, 6.3, 6.6 and 6.8). Of the tumours studied, 11.9% contained isoforms of pI 6.1, 6.6 and 6.8, with 14.3% containing isoforms with pI 6.1, 6.6 and 6.3. Very few tumours contained only one isoform, with 4.8% of tumours containing a single isoform at pI 6.1 and 2.4% of tumours containing only the isoform at pI 6.6.

All four ER isoforms were also shown to be present in some tumours by immunoblotting using antibody H222 and, in addition, high-resolution IEF indicated that all isoforms bind oestradiol, diethylstilboestrol and tamoxifen.

The variability in the ER isoform profile may have a bearing on the known variability of tumour response to endocrine therapy and prognosis.