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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
We have generated hybridomas which secrete monoclonal antibodies to the AT1 subtype of the angiotensin II receptor (AT1 receptor). These were obtained after immunization of Balb C/c mice with synthetic peptides representing sequences from either the extracellular domain (residues 8-17) or the intracellular domain (residues 229-237) of the AT1 receptor.
Hybridoma populations were first screened for the production of antibodies which bound to rat liver cells. Further selection, and cloning by limiting dilution, was carried out for antibodies which bound specifically to rat adrenal glomerulosa cells. Confirmation that the antibody designated 6313/G2 interacted with the angiotensin II receptor was obtained using COS-7 cells transfected with AT1A receptor cDNA. In particular, the initial characterization of 6313/G2 showed specific immunofluorescence of vascular endothelium.
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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.
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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.
