In animal models the somatostatin analog angiopeptin inhibits intimal hyperplasia by acting primarily through somatostatin receptor 2 (SSTR-2). However, the results of clinical trials using angiopeptin have been disappointing. In this study we showed that human blood vessels express high levels of SSTR-1 with significantly lower levels of SSTR-2 and -4. Samples of normal veins and arteries, as well as atherosclerotic arteries, expressed predominantly SSTR-1. In addition, the levels of SSTR-1 varied between individuals, indicating that the vascular disease process may have affected SSTR gene expression. Immunocytochemical studies demonstrated that SSTR-1 was present in endothelial but not vascular smooth muscle cells. No evidence of SSTR-3 or -5 expression was detected in normal or diseased blood vessels. Two endothelial cell preparations, ECV304 and human umbilical vein endothelial cells, were investigated and shown to express only SSTR-1 and -4. Exposure of these cells to 10 nM somatostatin or 10 nM SSTR-1-specific agonist resulted in alterations to the actin cytoskeleton, as characterized by a loss of actin stress fibers coupled with an increase in lamellipodia formation at the plasma membrane. These results suggest that the lack of effectiveness of angiopeptin in humans may be due to the differential expression of SSTR-1 by human endothelial cells.
The movement of urea across plasma membranes is modulated by facilitated urea transporter proteins. These proteins are the products of two closely related genes, termed UT-A (Slc14a2) and UT-B (Slc14a1). By genomic library screening and P1 artificial chromosome "shotgun" sequencing, we have determined the structure of the mouse UT-A gene. The gene is Ͼ300 kb in length, contains 24 exons, and has 2 distinct promoters. Flanking the 5Ј-region of the gene is the UT-A␣ promoter that regulates transcription of UT-A1 and UT-A3. The second promoter, termed UT-A, is present in intron 13 and regulates transcription of UT-A2. cAMP agonists (100 M dibutryl cAMP, 25 M forskolin, 0.5 mM IBMX) increased the activity of a 2.2-kb UT-A␣ promoter construct 6.2-fold [from 0.026 Ϯ 0.003 to 0.160 Ϯ 0.004, relative light units (RLU)/g protein] and a 2.4-kb UT-A promoter construct 9.5-fold (from 0.020 Ϯ 0.002 to 0.190 Ϯ 0.043 RLU/g protein) above that in untreated controls. Interestingly, only the UT-A promoter contained consensus sequences for CREs and deletion of these elements abolished cAMP sensitivity. Increasing the tonicity of culture medium from 300 to 600 mosmol/kgH2O with NaCl caused a significant increase (from 0.060 Ϯ 0.004 to 0.095 Ϯ 0.010 RLU/g protein) in UT-A␣ promoter activity but had no effect on the UT-A promoter. A tonicity-responsive enhancer was identified in UT-A␣ and is suggested to be responsible for mediating this effect. Levels of UT-A2 and UT-A3 mRNA were increased in thirsted mice compared with control animals, indicating that the activities of both promoters are likely to be elevated during prolonged antidiuresis.
Attachment of a cleavable hexa His tag is a common strategy for the production of recombinant proteins. Production of two recombinant nonglycosylated human serum transferrins (hTF-NG), containing a factor Xa cleavage site and a hexa His tag at the carboxyl terminus, has been described [Mason et al. (2001) Prot. Exp. Purif 23, 142-150]. More recently, hTF-NG with an amino-terminal His tag and a factor Xa cleavage site has been expressed (>30 mg/L) in baby hamster kidney cells and purified from the tissue culture medium. Although it is frequently assumed that addition of a His tag has little or no effect on function, this is not always confirmed experimentally. In the present study, in vitro quantitative data clearly shows that the presence of the C-terminal His tag has an effect on the release of iron from recombinant hTF at pH 7.4 and 5.6. Measurement of the rate of release from both the N- and C-lobes is reduced 2-4-fold. These findings provide further compelling evidence that the two lobes communicate with each other and highlight the importance of the C-terminal portion of the C-terminal lobe in this interaction. In contrast to these results, we demonstrate that the presence of a His tag at the N-terminus of hTF has no effect on the rate of iron release from either lobe. In a competition experiment, both unlabeled N- and C-terminal His-tagged constructs were equally effective at inhibiting the binding of radio-iodinated diferric glycosylated hTF from a commercial source to receptors on HeLa cells as the unlabeled recombinant diferric hTF-NG control. Thus, the presence of a His tag at either the N- or C-terminus of hTF-NG has no apparent effect on the ability of these hTF species to bind to transferrin receptors.
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