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ABSTRACT
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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ABSTRACT
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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ABSTRACT
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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ABSTRACT
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.
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ABSTRACT
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 (K d=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 (P<0·05) and kidney (P<0·01), while in liver the binding to the isoform of pI 6·7 was significantly reduced (P<0·001).
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.
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ABSTRACT
Using labelled ligand-binding methods, previous studies have identified specific angiotensin II receptors (Ang II-Rs) in eel liver, kidney and intestine membranes. Isoelectric focusing on polyacrylamide gels also showed that there are two Ang II-R isoforms in eel liver, focusing at isoelectric points (pI) 6·5 and 6·7. These may have different functions. In contrast, eel enterocyte plasma membrane and renal brush border membranes contain only the pI 6·5 form.
To characterize the eel receptors more fully, a newly developed monoclonal antibody (6313/G2) which selectively recognizes the AT1 subtype of mammalian Ang II-R was used. In ligand-binding experiments, the preincubation of eel liver membranes with 6313/G2 antibody eliminated the specific [3,5-3H]Tyr4-Ile5-Ang II binding. Moreover, Ang II—receptor complexes from solubilized liver membranes, which were immunoprecipitated by 6313/G2-coated beads, had a pI of 6·5. In immunoblotting experiments, the antibody recognized the isoform focusing at pI 6·5 in eel intestine and liver preparations, but not the liver pI 6·7 isoform. Immunoblotting of SDS gels showed that the antibody bound to a single protein of molecular mass of 75 kDa in eel liver, gill and kidney and to a doublet of molecular mass of about 74 and 75 kDa in intestinal membrane preparations. Immunocytochemistry of paraffin-embedded and cryostat sections of eel liver, kidney, intestine and gill showed that antibody 6313/G2 bound to uniformly distributed intracellular sites and cell surface membranes in proximal tubular cells, absorptive intestinal cells, hepatocytes and chloride cells. It also stained endothelium and both the longitudinal and circular layers of smooth muscle cells in the intestine.
The data suggest that the previously described Ang II-R from eel liver, kidney and intestine may be similar to the mammalian AT1 subtype.
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ABSTRACT
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.
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ABSTRACT
Oestrogen receptors (ERs) in breast tumours are highly heterogeneous. In previous studies we have shown that at least four isoforms may exist. These migrate in isoelectric focusing (IEF) gels to isoelectric points (pI values) 6·1, 6·3, 6·6 and 6·8. Of these the first (pI 6·1) corresponds to the 8S isoform as detected by sucrose gradient fractionation, while the others all sediment at 4S. In a series of 66 breast tumours it was found that those at pI 6·3 and pI 6·8 were significantly correlated with the presence of progesterone receptors.
To characterize the isoforms more fully, ER isoforms labelled by [3H]oestradiol binding were fractionated by IEF. The results were compared with those obtained after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using the H222 anti-ER monoclonal antibody. In other experiments, tumour ER isoforms were covalently labelled with [ring-3H] tamoxifen aziridine and separated by IEF. The individual isoforms were electroeluted from the IEF gel and further analysed by SDS-PAGE and non-denaturing PAGE. In summary, the evidence shows that the isoforms of pI values 6·3, 6·8 and 6·6 have molecular masses of 50, 65 and 70 kDa respectively. In addition, all three of these isoforms, i.e. the pI 6·3, 6·8 and 6·6 isoforms, could form dimers.
We conclude that the three isoforms sedimenting at 4S have the capacity to form dimers and thus may have the potential for binding to oestrogen response elements in the genome.
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ABSTRACT
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.
